The cGMP-gated channel of the rod photoreceptor cell characterization and orientation of the amino terminus.

The molecular properties and orientation of the cGMP-gated cation channel of bovine rod outer segment membranes were studied using biochemical and immunochemical methods. Western blots labeled with anti-channel monoclonal antibodies indicate that the channel has a subunit Mr of 63,000 in bovine rod outer segment membranes prepared in the presence and absence of protease inhibitors and in rod outer segments from other mammalian retinas. The channel has an apparent Mr of 78,000 in both COS-1 cells and Xenopus oocytes expressing the cloned cDNA. NH2-terminal sequence analysis indicates that the lower Mr of the channel in rod outer segments is caused by the absence of the first 92 amino acids predicted by cDNA sequence analysis. Immunofluorescent and immunogold labeling has confirmed that the 63,000 form of the channel is present in rod outer segments. These results indicate that photoreceptor cell-specific co-translational or post-translational cleavage of the NH2-terminal segment of the channel occurs prior or during the incorporation of the channel into the rod outer segment plasma membrane. Immunogold labeling studies using site-directed antibodies also indicate that the NH2-terminal segment of the rod outer segment channel is exposed on the cytoplasmic side of the plasma membrane.     

Metabolism of phosphatidylethanolamine in the frog retina

The synthesis and the turnover of phosphatidylethanolamine in frog retinal rod outer segments and microsomes were studied by monitoring the incorporation of five radioactive precursors: 32PO4, 33PO4 [3H]glycerol, [3H]serine, and [3H]ethanolamine. 1. Labeled serine was actively incorporated into phosphatidylethanolamine. The kinetics of the labeling patterns in both microsomes and rod outer segments was consistent with formation via decarboxylation of phosphatidylserine. 2. Ethanolamine was found to be an ineffective precursor of phosphatidylethanolamine, suggesting that the major pathway for phosphatidylethanolamine synthesis in the retina is via the decarboxylation reaction. 3. An active methylation of phosphatidylethanolamine to phosphatidylcholine was observed in both retinal microsomes and rod outer segments. 4. The kinetics of labeling of phosphatidylethanolamine in the rod outer segments was different for the various isotopic precursors, and was found to depend on the relative turnover times of the precursor pools. Glycerol was the only precursor that gave a true pulse of radioactivity. 5. The specific activity of phosphatidylethanolamine derived from labeled glycerol declined exponentially, demonstrating that the labeled lipid was diffusely distributed throughout the rod outer segments. The half-life of phosphatidylethanolamine in the rod outer segments was determined to be 18 days. Comparison of this value to the turnover time of rod outer segment integral proteins revealed that rod outer segment lipid is renewed at a faster rate than protein.     

Glyceraldehyde-3-phosphate dehydrogenase is a major protein associated with the plasma membrane of retinal photoreceptor outer segments

A major 38-kDa protein associated with bovine rod outer segment plasma membranes, but not disk membranes, has been identified as glyceraldehyde-3-phosphate dehydrogenase on the basis of its N-terminal sequence and specific enzyme activity. This enzyme was extracted from lysed rod outer segments or isolated rod outer segment plasma membrane with 0.15 M NaCl and purified to homogeneity by affinity chromatography on a NAD(+)-agarose column. A specific activity of 90-100 units/mg of protein is within the range of activity obtained for glyceraldehyde-3-phosphate dehydrogenase isolated from other mammalian cells. Enzyme activity measurements indicate that this enzyme makes up approximately 2% of the total rod outer segment protein and over 11% of the plasma membrane protein. Protease digestion and binding studies on purified rod outer segment plasma and disk membranes suggest that glyceraldehyde-3-phosphate dehydrogenase reversibly interacts with a protease-sensitive plasma membrane-specific protein of rod outer segments. The finding that glyceraldehyde-3-phosphate dehydrogenase is present in large quantities in rod outer segments suggests that at least some of the energy required for the synthesis of ATP and GTP for phototransduction and other processes of the outer segment is derived from glycolysis which takes place within this organelle.     

Histo- and cytochemical demonstration of guanosine triphosphatase activity in the outer segments of rods in the rat retina by means of a newly developed method

Summary Guanosine triphosphatase (GTPase) activity was studied histo- and cytochemically in the rod outer segments of the rat retina by means of a newly developed method. Differences in the distribution pattern of the enzyme activity exist within the outer segment: the activity is more intense at the tip of the rod outer segments near the pigment epithelium than in their proximal portion. Ultracytochemically, the new procedure reveals the reaction product of GTPase activity partly (i) on the extradisk membrane side and (ii) on the disk membranes. This result is in contrast to the cytochemical localization of guanylate cyclase (GCLase), an enzyme also localized at the tip of the rod outer segments: GCLase activity is restricted to the intradisk membrane area of the rod outer segments. The functional role of GTPase activity in the outer segments of rods is discussed.The authors dedicate this paper to Professor K. Ogawa     

Biochemical aspects of the visual process. XXV. Light-induced calcium movements in isolated frog rod outer segments

Suspensions of isolated rod outer segments are shown to have a high calcium content of up to 11 moles calcium per mole rhodopsin. Osmotic lysis of the outer segments demonstrates the presence of two calcium fractions, a soluble one and a particulate one. The particulate fraction apparently coincides with the rod disks or with disk fragments. Illumination of intact rod outer segments in calcium-free ATP-containing Ringer solution has no measurable effect upon their total caclium content, but causes a significant shift of calcium from the particulate to the soluble fraction. This light effect is retained in lysed outer segments resuspended in calcium-free ATP-containing Ringer. These results support a function of calcium as a transmitter in light transduction in rod outer segments.     

Molecular mechanisms of photoreception. IV. Ca2+-inhibited GTPase of rod outer segments of the frog retina

Ca2+-dependent GTPase activity is found to be present in the rod outer segments of frog retina. GTPase localization in rod outer segments is shown by fractionating the rod outer segment preparation in the sucrose density gradient. The enzyme is readily washed out of cells with isotonic NaCl solution. The Km is 0.6 mM for GTP. The activity is inhibited by 78 +/- 12% with the increase in Ca2+ concentration from 10(-9) to 10(-7) M. GTP hydrolysis is inhibited by the same concentrations of Ca2+ which block the sodium conductivity of the rod outer segment cytoplasmic membrane.     

Glycoproteins specific for the retinal rod outer segment plasma membrane

Two ricin-specific glycoproteins have been identified on neuraminidase-treated rod outer segment plasma membranes of bovine retinal photoreceptor cells. Ricin-gold-dextran particles were observed by electron microscopy to densely label the surface of neuraminidase-treated rod outer segments. Western blotting of proteins separated by SDS-gel electrophoresis indicated that two ricin-binding glycoproteins of Mr 230,000 and 110,000 are specific for the plasma membrane and are not found in disk membranes. These glycoproteins can serve as specific probes for the purification of the rod outer segment plasma membrane.     

The isolation of stable cattle rod outer segments with an intact plasma membrane.

A procedure is described to purify and stabilize cattle rod outer segments with an intact plasma membrane. Three criteria are applied to assess the integrity of the latter. Upon photolysis in these rod outer segments: (1) exogenous ATP cannot phosphorylate rhodopsin located in the disk membrane. (2) Endogenous cofactors (NADPH, NADPH-regenerating system) are still available in the rod cytosol and consequently retinol is the final photoproduct of photolysis of rhodopsin. (3) The rod cytosol can maintain a pH different from that of the medium, since the later stages of rhodopsin photolysis are independent of the medium pH. The stability and homogeneity of the preparation appear to be much better than those of freshly isolated frog rod outer segments, which have been used most frequently so far for experiments on the physiology of rod outer segments. In addition, these cattle rod outer segments remain intact during various manipulations and therefore considerably extend the experimental possibilities when intact rod outer segments are required.     

Immunocytochemical localization of taurine in the fish retina under light and dark adaptations

Summary. Previously we have observed the lack of immunoreactivity of taurine in the rod outer segments from light-adapted fish, such as the ayu Plecoglossus altivelis and lefteye flounder Paralichthys olivaceus. This finding prompted us to investigate if there is a difference in the immunocytochemical localization of taurine in the rod outer segments between the dark- and light-adapted states. In the retinas of the glass eel Anguilla japonica and the young goldfish Carassius auratus, extremely intense immunostaining was found in the cone outer segments, rod inner segments, photoreceptor supranuclear region and outer plexiform layer. The rod outer segments were not immunostained in the light-adapted state, while they were intensely immunostained in the dark-adapted state. Consequently, it was suggested that the lack of immunoreactivity in the rod outer segment may depend on light stimulation. In addition, the conspicuous immunocytochemical localization of taurine was discussed with the possible functional roles for taurine in the fish retina. Received January 25, 2000/Accepted January 31, 2000     

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.     

Correlation of rhodopsin biogenesis with ultrastructural morphogenesis in the chick retina

The developing chick retina from stages 39-45 has been examined by biochemical and electron microscope techniques. The levels of rhodopsin contained in the maturing chick retina were evaluated by detergent extraction and correlated with rod outer segment formation. It was found that the appearance of rhodopsin in significant levels preceded outer segment formation by at least 2 days, thus implying that rhodopsin is synthesized in the receptor cell inner segment and translocated to the outer limb when disk membrane biogenesis occurs. The level of rhodopsin continues to rise as the rod outer segment develops. Development of both rods and cones originates and proceeds most rapidly in the fundus or central region and proceeds toward the periphery. In general, rod outer segments were noted to develop far more rapidly than cone outer segments.     

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.     

The isolation of stable cattle rod outer segments with an intact plasma membrane

A procedure is described to purify and stabilize cattle rod outer segments with an intact plasma membrane. Three criteria are applied to assess the integrity of the latter.Upon photolysis in these rod outer segments: (1) exogenous ATP cannot phosphorylate rhodopsin located in the disk membrane. (2) Endogenous cofactors (NADPH, NADPH-regenerating system) are still available in the rod cytosol and consequently retinol is the final photoproduct of photolysis of rhodopsin. (3) The rod cytosol can maintain a pH different from that of the medium, since the later stages of rhodopsin photolysis are independent of the medium pH.The stability and homogeneity of the preparation appear to be much better than those of freshly isolated frog rod outer segments, which have been used most frequently so far for experiments on the physiology of rod outer segments. In addition, these cattle rod outer segments remain intact during various manipulations and therefore considerably extend the experimental possibilities when intact rod outer segments are required.     

Rod-to-Hook Transition for Extracellular Flagellum Assembly Is Catalyzed by the L-Ring-Dependent Rod Scaffold Removal

In Salmonella, the rod substructure of the flagellum is a periplasmic driveshaft that couples the torque generated by the basal body motor to the extracellular hook and filament. The rod subunits self-assemble, spanning the periplasmic space and stopping at the outer membrane when a mature length of ∼22 nm is reached. Assembly of the extracellular hook and filament follow rod completion. Hook initiation requires that a pore forms in the outer membrane and that the rod-capping protein, FlgJ, dislodges from the tip of the distal rod and is replaced with the hook-capping protein, FlgD. Approximately 26 FlgH subunits form the L-ring around the distal rod that creates the pore through which the growing flagellum will elongate from the cell body. The function of the L-ring in the mature flagellum is also thought to act as a bushing for the rotating rod. Work presented here demonstrates that, in addition to outer membrane pore formation, L-ring formation catalyzes the removal of the FlgJ rod cap. Rod cap removal allows the hook cap to assemble at the rod tip and results in the transition from rod completion in the periplasm to extracellular hook polymerization. By coupling the rod-to-hook switch to outer membrane penetration, FlgH ensures that hook and filament polymerization is initiated at the appropriate spatial and temporal point in flagellar biosynthesis.     

Anti-lamprey retinal antibodies: immunohistochemistry on the retinas of several species of vertebrates

Summary Two monoclonal antibodies, H16 and B11, which were raised against lamprey retinal homogenate, were found to react with both short and long photoreceptor outer segments. On Western blotting of the retinal homogenate, both antibodies recognized a 40000 Da and a 80000 Da band. H16 antibody stained rod outer segments of all examined vertebrates, all cone outer segments of the turtle and chicken, and certain cone outer segments of the macaque. B11 antibody stained sub-mammalian rod outer segments and some mammalian cone outer segments, leaving all mammalian rod outer segments unstained. The epitope recognized by H16 antibody is considered to be located in a conserved or commonly inherited element of an outer segment-bound molecule, presumably rhodopsin. B11 antibody, on the other hand, seems to recognize a reactive group which has failed to be inherited by mammalian rod cells; why it recognizes all cone outer segments in the turtle and chicken and only a part of them in the cow, cat, and macaque, meanwhile ignoring all of them in the frog and fish, is subject to further study.A portion of this work is contained in a dissertation submitted by the first author, M.Y., in application for the Ph.D. degree at Yamagata University School of Medicine.     

Characterization of the recombination reaction of rhodopsin.

The kinetics of recombination of 11-cis-retinal with bleached rod outer segments and sodium cholate solubilized rhodopsin have been investigated. At neutral pH, it was found that bleached rod outer segments in the presence of an excess of 11-cis-retinal follow pseudo-first-order kinetics. The results suggest the second-order formation of an intermediate addition compound followed by a first-order dehydration step to form a protonated aldimine linkage. In addition, at pH values above 7.5 or below 6.5 the kinetics of recombination are complex, indicating the formation of a molecular species inactive in recombination which is in equilibrium with the active form of opsin. Based upon the observed rate constants as a function of pH, a scheme is presented to describe the recombination reaction in bleached rod outer segments. The kinetics of recombination of sodium cholate solubilized opsin were also analyzed. In terms of formation of an intermediate addition compound and subsequent dehydration, the values for the individual rate constants for both bleached rod outer segments and cholate-solubilized opsin were found to compare very favorably. These results demonstrate that the sodium cholate (2 mg/ml) maintains opsin in a conformation very similar to that in the rod outer segment membrane and suggest that the cholate-opsin complex is an excellent model system for studies on opsin-membrane interactions.     

Conductance of cytoplasmic membrane of photoreceptors by the method of intracellular dialysis

To clear up the effect of agents proposed as a mediator in the phototransduction process on the rod outer segment cytoplasmic membrane a technique based on the intracellular dialysis method was developed. It was shown that in the presence of nucleoside-triphosphates 3',5'-cGMP increased cytoplasmic membrane sodium conductance of dialyzed rod outer segment by 1-4 nS. The dependence of the effect amplitude on the cyclic nucleotides concentrations was obtained. It is suggested that the conductance changes observed are due to the action of cyclic nucleotide dependent protein kinase on the rod outer segment plasma membrane.     

Rhodopsin in the rod outer segment plasma membrane

Isolated frog retinas were incubated in vitro with a 4-h pulse of [3H]leucine, then chased for 32 h with a nonradioactive amino acid mixture. At the end of the incubation, light and electron microscope autoradiograms were prepared from some of the retinas. The autoradiograms revealed: (a) intense radioactivity in the basal disks of the rod outer segments, (b) diffuse label evenly distributed throughout the rod outer segments, and (c) a high concentration of label in the entire rod outer segment plasma membrane. Incubation under identical conditions, but with puromycin added, significantly inhibited the labeling of all of these components. To identify the labeled proteins, purified outer segments from the remaining retinas were analyzed biochemically by SDS disc gel electrophoresis and gel filtration chromatography. SDS gel electrophoresis showed that about 90% of the total rod outer segment radioactivity chromatographed coincident with visual pigment, suggesting that the radiolabeled protein in the plasma membrane is visual pigment. Gel filtration chromatography demonstrated that the radiolabeled protein co-chromatographed with rhodopsin rather than opsin, and that the newly synthesized visual pigment is both the basal disks and the plasma membrane is present in the native configuration.     

Biochemical aspects of the visual process XXXII. Movement of sodium ions through bilayers composed of retinal and rod outer segment lipids

The leakage of Na⁺ from sonicated liposomes, composed of rod outer segment lipids, retinal lipids and a 4 : 1 phosphatidylcholine/phosphatidylserine mixture, has been studied. Both retinal and rod outer segment lipid liposomes lose Na⁺ faster than Ca²⁺ which indicates that the observed leakage occurs from closed liposomal structures.Liposomes from rod outer segment lipids are extremely leaky, losing sodium about 10 times as fast as retinal lipid liposomes and twice as fast as the phosphatidylcholine/phosphatidylserine liposomes.This high permeability of rod outer segment lipid liposomes, as compared to retinal lipid liposomes, is probably due to both the higher degree of unsaturation of the fatty acid chains and their lower cholesterol content. In the rod outer segment lipid extract 48% of the fatty acid chains consists of docosahexaenoic acid (C_22:6) against only 24% in retinal lipid extract. Rod outer segment lipids contain 4.0% cholesterol against 12.3% in retinal lipids.The sodium leakage from rod outer segment lipid liposomes is little affected by the presence of 5 mM calcium in the external dialysis medium, but with the two other types of liposomes significant decreases in permeability of about 20% are observed.The results are discussed in connection with the role of cations in visual excitation.     

Oxidative damage of retinal rod outer segment membranes and the role of vitamin E

Highly purified bovine rod outer segment membranes show loss of structural integrity under an air atmosphere. Obvious ultrastructural changes are preceded by increases in absorbance below 400 nm. These changes are inhibited by Ar or N₂ atmospheres and appear to be due primarily to oxidative damage to the polyunsaturated fatty acids of the membrane lipids. Loss of polyunsaturated fatty acids, formation of malonaldehyde and fluorescent products characteristic of lipid oxidation accompany the spectral alterations. The elevated ultraviolet absorbance can largely be removed from the membranes by gentle extraction of the lipids using phospholipase C and hexane without changing the visible absorbance of rhodopsin.We have found a large seasonal variation in the endogenous level of α-tocopherol (vitamin E) in the bovine rod outer segment preparations. For much of the year we find that the rod outer segment membranes contain higher levels of α-tocopherol than have been previously reported in biological membranes. Rod outer segments which are low in endogenous tocopherol can be protected from oxygen damage by adding exogenous tocopherol. The rod outer segments are extremely susceptible to oxygen damage due to the unusually high content of polyunsaturated fatty acids in the membrane lipids. The presence of tocopherol inhibits oxygen damage but does not eliminate it. The tocopherol in the rod outer segments is consumed in air, thus complete protection from peroxidation in vitro requires an inert atmosphere as well as high levels of tocopherol.This work suggests that extensive precautions against oxidative degradation should also be employed in studies of other membrane systems where important deleterious effects of oxygen may be less obvious.