The role of retinal photoisomerase in the visual cycle of the honeybee

The compound eye of the honeybee has previously been shown to contain a soluble retinal photoisomerase which, in vitro, is able to catalyze stereospecifically the photoconversion of all-trans retinal to 11-cis retinal. In this study we combine in vivo and in vitro techniques to demonstrate how the retinal photoisomerase is involved in the visual cycle, creating 11-cis retinal for the generation of visual pigment. Honeybees have approximately 2.5 pmol/eye of retinal associated with visual pigments, but larger amounts (4-12 pmol/eye) of both retinal and retinol bound to soluble proteins. When bees are dark adapted for 24 h or longer, greater than 80% of the endogenous retinal, mostly in the all-trans configuration, is associated with the retinal photoisomerase. On exposure to blue light the retinal is isomerized to 11-cis, which makes it available to an alcohol dehydrogenase. Most of it is then reduced to 11-cis retinol. The retinol is not esterified and remains associated with a soluble protein, serving as a reservoir of 11-cis retinoid available for renewal of visual pigment. Alternatively, 11-cis retinal can be transferred directly to opsin to regenerate rhodopsin, as shown by synthesis of rhodopsin in bleached frog rod outer segments. This retinaldehyde cycle from the honeybee is the third to be described. It appears very similar to the system in another group of arthropods, flies, and differs from the isomerization processes in vertebrates and cephalopod mollusks.     

The 3, 4-didehydroretinal chromophore of goldfish porphyropsin

The isomeric configuration of the 3,4-didehydroretinal chromophore of goldfish porphyropsin was determined by high performance liquid chromatography (HPLC) and by the regeneration of this visual pigment with authentic isomers of 3,4-didehydroretinal. A nonisomerizing, quantitative method using hydroxylamine and methylene chloride was employed to extract the 3,4-didehyroretinal chromophore from the rod outer segment membrane (containing the porphyropsin). When this extracted chromophore was injected into the HPLC, only a single major peak was observed and this peak coeluted with the authentic 11-cis 3,4-didehydroretinyl oxime. This suggests that the chromophore of goldfish porphyropsin is 11-cis 3,4-didehydroretinal. When the bleached rod outer segments (containing the opsin) were incubated with different 3,4-didehydroretinal isomers (13-cis, 11-cis, 9-cis, and all-trans), only the 11-cis isomer resulted in the degeneration of porphyropsin. This also suggests that the porphyropsin chromophore exists in the 11-cis configuration.     

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.     

Isomerization and oxidation of vitamin a in cone-dominant retinas: a novel pathway for visual-pigment regeneration in daylight

The first step toward light perception is 11-cis to all-trans photoisomerization of the retinaldehyde chromophore in a rod or cone opsin-pigment molecule. Light sensitivity of the opsin pigment is restored through a multistep pathway called the visual cycle, which effects all-trans to 11-cis re-isomerization of the retinoid chromophore. The maximum throughput of the known visual cycle, however, is too slow to explain sustained photosensitivity in bright light. Here, we demonstrate three novel enzymatic activities in cone-dominant ground-squirrel and chicken retinas: an all-trans-retinol isomerase, an 11-cis-retinyl-ester synthase, and an 11-cis-retinol dehydrogenase. Together these activities comprise a novel pathway that regenerates opsin photopigments at a rate 20-fold faster than the known visual cycle. We suggest that this pathway is responsible for sustained daylight vision in vertebrates.     

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.     

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.     

Biochemical aspects of the visual process. XXVIII. Classification of sulfhydryl groups in phodopsin and other photoreceptor membrane proteins.

Reaction of isolated bovine rod outer segment membrane with radioactive N-ethylmaleimide, both in the presence and absence of 1% dodecyl sulfate followed by dodecyl sulfate-polyacrylamide gel electrophoresis, shows that six sulfhydryl groups (96% of total sulfhydryl in this membrane) are located on the rhodopsin molecule. On the basis of their reactivity towards rho-chloromercuribenzoate and rho-chloromercuribenzene sulfonate in suspensions of outer segment membranes, the sulfhydryl groups of rhodopsin can be divided into three pairs. One pair is rapidly modified, both in light and darkness. This modification does not impair the recombination capacity of opsin with 11-cis retinaldehyde under regeneration of rhodopsin. A second pair is modified upon prolonged interaction with the rho-chloromercuriderivatives in darkness. Modification of this pair leaves the typical rhodopsin absorbance at 500 nm intact, but a proportional loss of recombination capacity does occur. The third pair is only modified after illumination and isprobably located in the vicinity of the chromophoric center. The differences between these results and those obtained by modification with dithiobis-(2-nitrobenzoic acid) or N-ethylmaleimide in suspension, where even upon prolonged exposure to light as well as in darkness only two sulfhydryl groups of rhodopsin are modified, is explained by the detergent-like character of the rho-chloromercuri-derivatives.     

A functional rhodopsin-green fluorescent protein fusion protein localizes correctly in transgenic Xenopus laevis retinal rods and is expressed in a time-dependent pattern

To study rhodopsin biosynthesis and transport in vivo, we engineered a fusion protein (rho-GFP) of bovine rhodopsin (rho) and green fluorescent protein (GFP). rho-GFP expressed in COS-1 cells bound 11-cis retinal, generating a pigment with spectral properties of rhodopsin (A(max) at 500 nm) and GFP (A(max) at 488 nm). rho-GFP activated transducin at 50% of the wild-type activity, whereas phosphorylation of rho-GFP by rhodopsin kinase was 10% of wild-type levels. We expressed rho-GFP in the rod photoreceptors of Xenopus laevis using the X. laevis principal opsin promoter. Like rhodopsin, rho-GFP localized to rod outer segments, indicating that rho-GFP was recognized by membrane transport mechanisms. In contrast, a rho-GFP variant lacking the C-terminal outer segment localization signal distributed to both outer and inner segment membranes. Confocal microscopy of transgenic retinas revealed that transgene expression levels varied between cells, an effect that is probably analogous to position-effect variegation. Furthermore, rho-GFP concentrations varied along the length of individual rods, indicating that expression levels varied within single cells on a daily or hourly basis. These results have implications for transgenic models of retinal degeneration and mechanisms of position-effect variegation and demonstrate the utility of rho-GFP as a probe for rhodopsin transport and temporal regulation of promoter function.     

THE ROLE OF THE PIGMENT EPITHELIUM IN THE ETIOLOGY OF INHERITED RETINAL DYSTROPHY IN THE RAT

Visual cell outer segment renewal was studied in eyes of mutant Royal College of Surgeons (RCS) and Sprague-Dawley (control) rats by a combination of microscopy and radioautography with the light and electron microscopes. RCS and control rats were injected with amino acids-³H at 11 days of age. Radioactive rod outer segment discs were assembled at the outer segment base from radioactive proteins synthesized in the rod inner segments. In controls, all radioactive discs assembled at 11 days of age were displaced the length of the outer segments, removed from outer segment tips, and phagocytized by the pigment epithelium by 8 days after injection. In the RCS rats, disc assembly and displacement resembled controls for the first 3 days after injection. However, as disc assembly continued for some time thereafter, a layer of labeled, disorganized, lamellar debris accumulated between the outer segment tips and the pigment epithelium. The buildup of debris was accompanied by visual cell death. At no time during the study was there evidence for phagocytic activity by the pigment epithelium. 61 days after injection, the layer of debris was the only heavily radioactive component in the retina. In the retina of RCS rats, the outer segment renewal mechanism malfunctions because the pigment epithelium does not fulfill its normal phagocytic role. The end result is visual cell death and blindness.     

Effect of 11-cis 13-demethylretinal on phototransduction in bleach-adapted rod and cone photoreceptors

We used 11-cis 13-demethylretinal to examine the physiological consequences of retinal's noncovalent interaction with opsin in intact rod and cone photoreceptors during visual pigment regeneration. 11-Cis 13-demethylretinal is an analog of 11-cis retinal in which the 13 position methyl group has been removed. Biochemical experiments have shown that it is capable of binding in the chromophore pocket of opsin, forming a Schiff-base linkage with the protein to produce a pigment, but at a much slower rate than the native 11-cis retinal (Nelson, R., J. Kim deReil, and A. Kropf. 1970. Proc. Nat. Acad. Sci. USA. 66:531-538). Experimentally, this slow rate of pigment formation should allow separate physiological examination of the effects of the initial binding of retinal in the pocket and the subsequent formation of the protonated Schiff-base linkage. Currents from solitary rods and cones from the tiger salamander were recorded in darkness before and after bleaching and then after exposure to 11-cis 13-demethylretinal. In bleach-adapted rods, 11-cis 13-demethylretinal caused transient activation of phototransduction, as evidenced by a decrease of the dark current and sensitivity, acceleration of the dim flash responses, and activation of cGMP phosphodiesterase and guanylyl cyclase. The steady state of phototransduction activity was still higher than that of the bleach-adapted rod. In contrast, exposure of bleach-adapted cones to 11-cis 13-demethylretinal resulted in an immediate deactivation of transduction as measured by the same parameters. These results extend the validity of a model for the effects of the noncovalent binding of a retinoid in the chromophore pockets of rod and cone opsins to analogs capable of forming a Schiff-base and imply that the noncovalent binding by itself may play a role for the dark adaptation of photoreceptors.     

Thermal activation and photoactivation of visual pigments

A visual pigment molecule in a retinal photoreceptor cell can be activated not only by absorption of a photon but also "spontaneously" by thermal energy. Current estimates of the activation energies for these two processes in vertebrate rod and cone pigments are on the order of 40-50 kcal/mol for activation by light and 20-25 kcal/mol for activation by heat, which has forced the conclusion that the two follow quite different molecular routes. It is shown here that the latter estimates, derived from the temperature dependence of the rate of pigment-initiated "dark events" in rods, depend on the unrealistic assumption that thermal activation of a complex molecule like rhodopsin (or even its 11-cis retinaldehyde chromophore) happens through a simple process, somewhat like the collision of gas molecules. When the internal energy present in the many vibrational modes of the molecule is taken into account, the thermal energy distribution of the molecules cannot be described by Boltzmann statistics, and conventional Arrhenius analysis gives incorrect estimates for the energy barrier. When the Boltzmann distribution is replaced by one derived by Hinshelwood for complex molecules with many vibrational modes, the same experimental data become consistent with thermal activation energies that are close to or even equal to the photoactivation energies. Thus activation by light and by heat may in fact follow the same molecular route, starting with 11-cis to all-trans isomerization of the chromophore in the native (resting) configuration of the opsin. Most importantly, the same model correctly predicts the empirical correlation between the wavelength of maximum absorbance and the rate of thermal activation in the whole set of visual pigments studied.     

A comparison of some photoreceptor characteristics in the pineal and retina

A rod-specific antiserum was used to immunolabel elements within the retina and pineal of the adult Djungarian hamster and Welsh Mountain sheep. In the retina immunostaining was localized to the outer segments and perikarya of photoreceptor cells, while in the pineal limited numbers of labelled pinealocytes were scattered throughout the gland. An enzyme-linked immunosorbent assay (ELISA) was then used to obtain a quantitative measure of rod opsin in total eye and pineal extracts from the Djungarian hamster. Total rod opsin (+/- SEM) in the eye was measured by absorbance spectroscopy (1.88 +/- 0.10 nmoles opsin/eye) and by using the ELISA (1.75 +/- 0.02 nmoles opsin/eye). The opsin content from a total of 56 pineals gave a mean value of 0.34 +/- 0.01 pmoles opsin/pineal. Since a functional photopigment should be coupled in a 1:1 ratio to a chromophore, we investigated whether we could identify 11-cis and/or all-trans retinaldehydes in the pineal extracts by quantitative extraction and HPLC analysis as the oximes. No evidence of 11-cis or all-trans retinaloxime could be found, the chromatograms were indistinguishable from those produced by extracts of cortical brain tissue. We conclude that the opsin present within the adult hamster pineal is not coupled to the common vertebrate retinaldehyde chromophore, and as a result, is unlikely to be part of a functional photopigment.     

The cGMP-gated channel and related glutamic acid-rich proteins interact with peripherin-2 at the rim region of rod photoreceptor disc membranes

The rod cGMP-gated channel is localized in the plasma membrane of rod photoreceptor outer segments, where it plays a central role in phototransduction. It consists of alpha- and beta-subunits that assemble into a heterotetrameric protein. Each subunit contains structural features characteristic of nucleotide-gated channels, including a cGMP-binding domain, multiple membrane-spanning segments, and a pore region. In addition, the beta-subunit has a large glutamic acid- and proline-rich region called GARP that is also expressed as two soluble protein variants. Using monoclonal antibodies in conjunction with immunoprecipitation, cross-linking, and electrophoretic techniques, we show that the cGMP-gated channel associates with the Na/Ca-K exchanger in the rod outer segment plasma membrane. This complex and soluble GARP proteins also interact with peripherin-2 oligomers in the rim region of outer segment disc membranes. These results suggest that channel/peripherin protein interactions mediated by the GARP part of the channel beta-subunit play a role in connecting the rim region of discs to the plasma membrane and in anchoring the channel.exchanger complex in the rod outer segment plasma membrane.     

A soluble form of bovine rod photoreceptor phosphodiesterase has a novel 15-kDa subunit

A substantial fraction (20-30%) of the bovine rod outer segment phosphodiesterase (PDE) activity is not associated with outer segment membranes prepared with buffers of moderate ionic strength; this PDE activity appears to represent a distinct, soluble isozyme. Although this PDE isozyme can be demonstrated to be present in sealed rod outer segments, it is discarded from most standard rod outer segment preparations. A method was developed that allowed the rapid purification of the soluble rod PDE by 2600-fold, to apparent homogeneity, using a monoclonal antibody column (ROS-1a). The soluble rod PDE isozyme has a novel Mr = 15,000 subunit (delta) in addition to subunits of Mr = 88,000 (alpha sol), 84,000 (beta sol), and 11,000 (gamma sol). The delta subunit comigrates with and may be identical to the cone PDE 15-kDa subunit. The small subunits of the soluble rod PDE and the membrane-associated rod PDE were isolated by reverse-phase chromatography. The gamma sol subunit was a potent inhibitor of trypsin-activated rod PDE, inhibiting 50% of 1 pM PDE activity at a concentration of 11 pM. This concentration was similar to that observed for the gamma subunit of the membrane-associated rod PDE. The purified delta subunit did not appear to affect PDE activity; this subunit was, however, unusually difficult to keep in solution. All of the kinetic and physical properties of the soluble rod PDE tested thus far are similar to those of the membrane-associated form, except for the presence of the delta subunit, suggesting that this unique subunit could mediate the solubility of the soluble rod PDE and the cone PDE in the intact photoreceptor.     

The effect of inhibitors of glycoprotein synthesis and processing on the phagocytosis of rod outer segments by cultured retinal pigment epithelial cells

Retinal pigment epithelial cells selectively phagocytize rod outer segments by a process that may be mediated by specific cell surface receptors. Since many receptors are glycoproteins, we have studied the effect of tunicamycin, an inhibitor of N-linked oligosaccharide synthesis, and of castanospermine and swainsonine, which are inhibitors of oligosaccharide processing, on the ability of cultured retinal pigment epithelial cells to phagocytize rod outer segment. Tunicamycin inhibits the glycosylation of newly synthesized glycoproteins by 85-90%; concomitantly, the phagocytosis of rod outer segments is inhibited by 70-80%. The effect of tunicamycin is to initially reduce rod outer segments binding, and therefore the subsequent ingestion of rod outer segments. SDS-PAGE analysis and autoradiography of [35S]methionine labelled extracts of tunicamycin-treated cells, demonstrates the disappearance of a number of glycoprotein bands, and the appearance of a number of protein bands of lower Mr. Kinetic analysis of the disappearance and reappearance of specific glycoproteins suggests that the lower Mr bands are the non-glycosylated forms of the higher Mr bands. By contrast, castanospermine and swainsonine have no effect on the ability of retinal pigment epithelial cells to phagocytize rod outer segments, or on the SDS-PAGE pattern of treated cells, although they were shown to inhibit oligosaccharide processing as expected. These results support the hypothesis that rod outer segment phagocytosis by retinal pigment epithelial cells is mediated by specific glycoprotein receptors. N-Glycosylation of these receptors is required for their function, or for their insertion into the plasma membrane, whereas processing of the N-linked oligosaccharide chains of these receptors is not crucial for rod outer segment phagocytosis by retinal pigment epithelial cells.     

Cyclic nucleotide-gated ion channels in rod photoreceptors are protected from retinoid inhibition

In vertebrate rods, photoisomerization of the 11-cis retinal chromophore of rhodopsin to the all-trans conformation initiates a biochemical cascade that closes cGMP-gated channels and hyperpolarizes the cell. All-trans retinal is reduced to retinol and then removed to the pigment epithelium. The pigment epithelium supplies fresh 11-cis retinal to regenerate rhodopsin. The recent discovery that tens of nanomolar retinal inhibits cloned cGMP-gated channels at low [cGMP] raised the question of whether retinoid traffic across the plasma membrane of the rod might participate in the signaling of light. Native channels in excised patches from rods were very sensitive to retinoid inhibition. Perfusion of intact rods with exogenous 9- or 11-cis retinal closed cGMP-gated channels but required higher than expected concentrations. Channels reopened after perfusing the rod with cellular retinoid binding protein II. PDE activity, flash response kinetics, and relative sensitivity were unchanged, ruling out pharmacological activation of the phototransduction cascade. Bleaching of rhodopsin to create all-trans retinal and retinol inside the rod did not produce any measurable channel inhibition. Exposure of a bleached rod to 9- or 11-cis retinal did not elicit channel inhibition during the period of rhodopsin regeneration. Microspectrophotometric measurements showed that exogenous 9- or 11-cis retinal rapidly cross the plasma membrane of bleached rods and regenerate their rhodopsin. Although dark-adapted rods could also take up large quantities of 9-cis retinal, which they converted to retinol, the time course was slow. Apparently cGMP-gated channels in intact rods are protected from the inhibitory effects of retinoids that cross the plasma membrane by a large-capacity buffer. Opsin, with its chromophore binding pocket occupied (rhodopsin) or vacant, may be an important component. Exceptionally high retinoid levels, e.g., associated with some retinal degenerations, could overcome the buffer, however, and impair sensitivity or delay the recovery after exposure to bright light.     

All-trans retinol in rod photoreceptor outer segments moves unrestrictedly by passive diffusion

The visual pigment protein of vertebrate rod photoreceptors, rhodopsin, contains an 11-cis retinyl moiety that is isomerized to all-trans upon light absorption. Subsequently, all-trans retinal is released from the protein and reduced to all-trans retinol, the first step in the recycling of rhodopsin's chromophore group through the series of reactions that constitute the visual cycle. The concentration of all-trans retinol in photoreceptor outer segments can be monitored from its fluorescence. We have used two-photon excitation (720 nm) of retinol fluorescence and fluorescence recovery after photobleaching to characterize the mobility of all-trans retinol in frog photoreceptor outer segments. Retinol produced after rhodopsin bleaching moved laterally in the disk membrane bilayer with an apparent diffusion coefficient of 2.5 +/- 0.3 micro m(2) s(-1). The diffusion coefficient of exogenously added retinol was 3.2 +/- 0.5 micro m(2) s(-1). These diffusion coefficients are in close agreement with those reported for lipids, suggesting that retinol is not tightly bound to protein sites that would be diffusing much more slowly in the plane of the membrane. In agreement with this interpretation, a fluorescent-labeled C-16 fatty acid diffused laterally with a similar diffusion coefficient, 2.2 +/- 0.2 micro m(2) s(-1). Retinol also moved along the length of the rod outer segment, with an apparent diffusion coefficient of 0.07 +/- 0.01 micro m(2) s(-1), again suggesting that it is not tightly bound to proteins that would confine it to the disks. The axial diffusion coefficient of exogenously added retinol was 0.05 +/- 0.01 micro m(2) s(-1). In agreement with passive diffusion, the rate of axial movement was inversely proportional to the square of the length of the rod outer segment. Diffusion of retinol on the plasma membrane of the outer segment can readily account for the measured value of the axial diffusion coefficient, as the plasma membrane comprises approximately 1% of the total outer-segment membrane. The values of both the lateral and axial diffusion coefficients are consistent with most of the all-trans retinol in the outer segments moving unrestricted and not being bound to carrier proteins. Therefore, and in contrast to other steps of the visual cycle, there does not appear to be any specialized processing for all-trans retinol within the rod outer segment.     

PARTICIPATION OF THE RETINAL PIGMENT EPITHELIUM IN THE ROD OUTER SEGMENT RENEWAL PROCESS

The disposal phase of the retinal rod outer segment renewal process has been studied by radioautography in adult frogs injected with tritiated amino acids. Shortly after injection, newly formed radioactive protein is incorporated into disc membranes which are assembled at the base of the rod outer segment. During the following 2 months, these labeled discs are progressively displaced along the outer segment owing to the repeated formation of newer discs. When the labeled membranes reach the end of the outer segment, they are detached from it. They subsequently may be identified in inclusion bodies within the pigment epithelium by virtue of their content of radioactivity. These inclusions have been termed phagosomes. Disc membrane formation is a continuous process, but the detachment of groups of discs occurs intermittently. The detached outer segment fragments become deformed, compacted, undergo chemical changes, and are displaced within the pigment epithelium. Ultimately, the material contained in the phagosomes is eliminated from the cell. In this manner the pigment epithelium participates actively in the disposal phase of the rod outer segment renewal process.     

Measurement of fast light-induced disc shrinkage within bovine rod outer segments by means of a light-scattering transient

A fast light-induced light-scattering transient, previously found in rod outer segment suspension, the so-called P-signal (Hofmann, K.P., Uhl, R., Hoffmann, W. and Kreutz, W. (1976) Biophys. Struct. Mechanism 2, 61–77), is described in more detail.The effect has the same action spectrum as rhodopsin bleaching. It is not regenerated with 11-cis retinal.The response is not linear with light-intensity for flashes which bleach more than 2.0% of rhodopsin; it saturates at an intensity corresponding to 15% rhodopsin bleaching.The wavelength- and scattering angle dependence lead to the conclusion that the change in light-scattering reflects a shrinkage of an osmotic compartment of the rod outer segment.The only compartment which we found to be intact in our rod outer segment preparations was the disc or rod sac; therefore, the effect must be attributed to a light-induced shrinkage of the rhodopsin-containing disc organelles.The overall effect (15% of rhodopsin is bleached) is in the range of 0.5–1.5% of the original volume.A light-induced passive cation-efflux from the disc, e.g. of Ca²⁺, can be ruled out as a possible molecular origin of the disc-shrinkage in our preparations.     

Occupancy of the chromophore binding site of opsin activates visual transduction in rod photoreceptors

The retinal analogue beta-ionone was used to investigate possible physiological effects of the noncovalent interaction between rod opsin and its chromophore 11-cis retinal. Isolated salamander rod photoreceptors were exposed to bright light that bleached a significant fraction of their pigment, were allowed to recover to a steady state, and then were exposed to beta-ionone. Our experiments show that in bleach-adapted rods beta-ionone causes a decrease in light sensitivity and dark current and an acceleration of the dim flash photoresponse and the rate constants of guanylyl cyclase and cGMP phosphodiesterase. Together, these observations indicate that in bleach-adapted rods beta-ionone activates phototransduction in the dark. Control experiments showed no effect of beta-ionone in either fully dark-adapted or background light-adapted cells, indicating direct interaction of beta-ionone with the free opsin produced by bleaching. We speculate that beta-ionone binds specifically in the chromophore pocket of opsin to produce a complex that is more catalytically potent than free opsin alone. We hypothesize that a similar reaction may occur in the intact retina during pigment regeneration. We propose a model of rod pigment regeneration in which binding of 11-cis retinal to opsin leads to activation of the complex accompanied by a decrease in light sensitivity. The subsequent covalent attachment of retinal to opsin completely inactivates opsin and leads to the recovery of sensitivity. Our findings resolve the conflict between biochemical and physiological data concerning the effect of the occupancy of the chromophore binding site on the catalytic potency of opsin. We show that binding of beta-ionone to rod opsin produces effects opposite to its previously described effects on cone opsin. We propose that this distinction is due to a fundamental difference in the interaction of rod and cone opsins with retinal, which may have implications for the different physiology of the two types of photoreceptors.