Opsin localization and glycosylation in the developing BALB/c and rds mouse retina.
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Animals↗
Biomedical subjects
D Bok
Publications and source records attributed to D Bok.
Light-activated retinoid transport in cephalopod photoreceptors.
Retinoid transport and chromophore exchange have been investigated in cephalopods using autoradiographic and radiobiochemical techniques. In dark adapted retinas, [3H]-retinoid is concentrated in myeloid bodies present in the photoreceptor inner segments and is bound to the photopigment retinochrome. In retinas exposed to light, there is a shift in the distribution of [3H]-retinoid. The rhabdomes become more heavily labeled than the inner segments, and rhodopsin labeling exceeds that of retinochrome. In animals returned to the dark, another shift in retinoid distribution occurs and the inner segments are again more labeled than the rhabdomes. In these animals [3H]-retinoid is bound primarily to retinochrome. Exposure to light seems to activate a transport mechanism that results in the redistribution of retinoid between the inner segments and rhabdomes and chromophore exchange among the photopigments.
Animals↗
Membrane receptors for retinol-binding protein in cultured human retinal pigment epithelium.
The retinal pigment epithelium (RPE) is responsible for transport of retinol from the choroidal circulation to the photoreceptors. In the intact eye, this process is mediated by membrane receptors for plasma retinol binding protein (RBP) distributed basolaterally on the RPE cells. We have shown that cultured human RPE expresses this receptor. A binding curve exhibiting saturation was generated by incubating enzymatically detached epithelial sheets with increasing concentrations of 125I-labelled RBP. 125I-RBP binding experiments also show that the receptor is expressed at a high level in first passage subcultures, suggesting de novo synthesis, and that basally oriented receptors predominate over those associated with the apical surface, reflecting the polarization characteristic of RPE in vivo. Cultured RPE can internalize 3H-retinol carried by RBP, resulting in synthesis of labelled retinyl palmitate. Production of labelled retinyl ester is competitively inhibited when incubations include an excess of holo-RBP containing non-radioactive retinol. These results indicate that RBP not only binds to the receptor specifically, but also that this interaction is functional, effecting uptake of retinol by the RPE cells. The expression of this property of differentiated RPE favors the use of cultured RPE as a model system for studying vitamin A transport and metabolism.
Adolescent↗
Melatonin-binding in the frog retina: autoradiographic and biochemical analysis.
Binding of melatonin was examined in the retina of Rana pipiens. When intact frog retinas were incubated with 3H-melatonin and processed for autoradiography, most of the radioactivity was localized to the melanosomes of the retinal pigment epithelium-choroid (RPE-choroid) and to the outer plexiform layer of the retina. Melanosome-enriched fractions of the RPE-choroid and membrane-enriched fractions of the neural retina demonstrated saturable melatonin binding when incubated with increasing melatonin concentration. Thin-layer chromatography showed that greater than 98% of the bound radioactivity was authentic melatonin. Scatchard analysis revealed a single population of binding sites with apparent Kd values of 6 X 10(-7) M for both the RPE-choroid and neural retina. When various indole analogs were tested for their ability to inhibit 3H-melatonin binding to the neural retina, both 5-methoxytryptophol and 6-chloromelatonin demonstrated complete displacement of melatonin binding. Endogenous retinal melatonin levels were measured by radioimmunoassay. A twofold increase in melatonin levels was observed during the dark period with peak levels at 384.5 +/- 28.8 pgms melatonin/pair retinas. Melatonin levels persisted in constant darkness, but were suppressed in constant light. Our data suggest that in the frog, the sites of action of retinal melatonin are the melanosomes of the RPE-choroid and the outer plexiform layer of the neural retina.
Animals↗
Immunoferritin localization of actin in retinal photoreceptors.
The contractile protein actin was recently localized to the distal portion of the connecting cilium in frog photoreceptors (Chaitin et al J Cell Biol 99:239-247, 1984). This is the site where the ciliary plasma membrane evaginates to form new outer segment disks (Steinberg et al J Comp Neurol 190: 501-518, 1980). In the present study, aldehyde fixed mammalian retinas were embedded in Lowicryl K4M, and thin tissue sections were reacted with antiactin antibodies using indirect immunoferritin labeling. Utilizing this technique, actin has been localized to the distal cilium in rat, cow, monkey, and human photoreceptors. These results provide additional evidence that an actin mediated contractile mechanism may regulate outer segment disk morphogenesis in vertebrate photoreceptors. As previously noted in frog retina, antiactin also labeled the bundle of filaments within photoreceptor calycal processes, and this label extended into the inner segment, subjacent to the plasma membrane. Within the inner segment, however, the striated rootlet was unlabeled.
Actins↗
The distribution of the main intrinsic membrane polypeptide in ocular lens.
The Main Intrinsic Polypeptide (MIP) of the ocular lens fiber cell plasma membrane was immunocytochemically localized at the ultrastructural level on ultrathin frozen sections of rat lens, and on extracted, gradient-purified bovine lens membranes. The results indicate that both the junctional and non-junctional membrane domains of the cortical lens fiber cell are MIP immunoreactive. Frozen thin section immunocytochemistry of the lens epithelium and hepatocytes, also using anti-MIP antibodies, revealed that these cells, and their intercellular junctions, are not MIP-immunoreactive. From these findings we conclude that 1) MIP, a putative fiber cell junctional protein, is present throughout the plasma membrane of the lens fiber cell, and is not confined to the fiber cell junctional domain, 2) MIP is not a detectable component of the lens epithelial cell membrane, or its intercellular junctions, 3) MIP is not detectable in gap junctions of hepatocytes.
Animals↗
Retinal photoreceptor-pigment epithelium interactions. Friedenwald lecture.
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Animals↗
Distribution patterns of photoreceptors, protein, and cyclic nucleotides in the human retina.
The concentration of cGMP, cAMP, protein and the number of cone and rod photoreceptors have been measured in parallel arrays of punches, 3 mm in diameter, taken from each quadrant of normal human retinas. A separate punch containing the fovea and parafoveal region was also analyzed. Eyes were obtained from four male donors ranging in age from 35 to 67 yr. The retina thins considerably from the center to the periphery, and consequently the protein content forms a gradient in the same direction. Similar gradients were observed for cAMP and cGMP concentrations. In all eyes studied, the foveal-parafoveal region had higher levels of cAMP than cGMP. The data was analyzed with the aid of a computer in order to obtain three-dimensional maps of the patterns of distribution of the different parameters. A strong correlation between the areas of higher cone density, non-photoreceptor neurons, and cAMP, and an equally strong correlation between rod distribution and that of cGMP was observed. These maps will serve as baseline data in studies of pathological conditions such as retinitis pigmentosa.
Adult↗
Localization of hydroxyindole-O-methyltransferase in the mammalian pineal gland and retina.
The pineal hormone, melatonin, has been reported to be synthesized in the retina by the enzyme, hydroxyindole-O-methyltransferase (HIOMT). Several laboratories have suggested that melatonin may be involved in photoreceptor outer segment disc shedding, photomechanical movements, and neuromodulation, but the cellular location of the retinal synthesizing enzymes has not been determined yet. Antiserum to HIOMT was obtained from rabbits immunized with bovine pineal extract. The monospecific immunoglobulins to HIOMT were isolated by positive-negative selection using pineal extract-sepharose and brain extract-sepharose affinity chromatography. The purity and specificity of the antibody to HIOMT was confirmed by immunodiffusion, electroblot immunolabeling, SDS-PAGE, and immunoprecipitin titration. Using the peroxidase-antiperoxidase (PAP) technique, HIOMT was localized in the pinealocytes of bovine and human pineal glands obtained during the light period. Rat pineal glands obtained during the dark period exhibited HIOMT immunoreactivity, whereas rat pineal glands obtained during the light period did not. Some pinealocytes of the bovine pineal did not exhibit HIOMT immunoreactivity, suggesting that not all pinealocytes are actively involved in melatonin synthesis. HIOMT was localized in the photoreceptors of bovine, rat, and human retinas, and some labeling also was observed in the inner retina, although the latter showed some species variation. This observation supports the hypothesis that photoreceptors are capable of melatonin synthesis.
Acetylserotonin O-Methyltransferase↗
Dietary restriction retards age-related loss of gamma crystallins in the mouse lens.
The soluble crystallins in lenses from diet-restricted and control mice of diverse ages (2, 11, or 30 months) were studied by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results obtained with both methods suggest that dietary restriction decelerates age-related loss of soluble gamma crystallins.
Aging↗
Autoradiographic and biochemical analysis of photoreceptor membrane renewal in Octopus retina.
Using autoradiographic and biochemical methods, we have demonstrated the renewal of light-sensitive membranes and photopigments in Octopus visual cells. After the injection of Octopus with [3H]leucine, electron microscope autoradiography revealed an intracellular pathway similar to that in vertebrates for the synthesis and transport of nascent protein from the inner segments to the rhabdomes. However, migration of labelled protein from synthetic sites to the light-sensitive rhabdomes took longer in Octopus than the equivalent process in vertebrates. Biochemical analysis of [3H]leucine-labelled retinas identified some of the labelled protein observed in autoradiographs of the rhabdomes as the visual pigment, rhodopsin. We have shown that retinochrome, a second photopigment in cephalopod retinas, is also renewed. Biochemical analysis 8 h after injection of [3H]leucine revealed heavy labelling of this photoprotein. Light microscope autoradiography of Octopus retina 8 h after injection of [3H]retinol showed labelling of both the rhabdomes and the myeloid bodies of the inner segments. Biochemical data gathered 8 h after injection of [3H]retinol indicated chromophore addition to both rhodopsin and retinochrome with retinochrome being more heavily labelled than rhodopsin. Thus, silver grains observed over the rhabdomes and inner segments could arise from one or both photopigments. These data suggest that retinal is stored in the myeloid bodies of the photoreceptor inner segments. Retinal could then be transferred, perhaps via retinochrome, to newly synthesized opsin before the visual pigment is assembled into new rhabdomeric membranes. Alternatively, retinochrome may serve to transport retinal from the myeloid bodies to the rhabdomes to regenerate rhodopsin as previously proposed.
Animals↗
Diurnal variations in amino acid incorporation into inner segment opsin.
Polyacrylamide gel and immunoelectrophoresis were used to measure the specific activity of nascent inner segment opsin following injections of labeled amino acids into frogs at different times during the diurnal cycle. Animals injected just after light onset showed the greatest incorporation of label into opsin, while animals injected during the night period had the lowest levels of isotope incorporation. As determined by rocket immunoelectrophoresis, there was a 13-fold decline in specific activity in animals injected during the late night hours in comparison with animals injected just after light onset. In contrast, rod outer segment rhodopsin-specific activity did not vary as greatly. Animals subjected to a 12-hour light-12-hour dark diurnal cycle incorporated only twice the radiolabeled amino acids into their rhodopsin compared with animals injected and maintained in constant dark for 24 hours. Taken together, these experiments suggest that adult Rana pipiens accumulate opsin within their inner segments during the dark phase of the diurnal cycle and that alterations of this pool effect changes in inner segment opsin specific activity. Differences in outer segment rhodopsin specific activity are probably due to averaged metabolic differences between constant dark and light-dark animals.
Absorption↗
Needed: a new way to train doctors (1).
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Curriculum↗
Needed: a new way to train doctors (2).
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Education, Medical↗
Blood vascular abnormalities in the degenerative mouse retina (C57BL/6J-rd le).
The authors have used a light microscopic horseradish peroxidase technique to demonstrate the arborization of blood vessels in mice homozygous for retinal degeneration and their normal heterozygous littermates. The results indicate a paucity of blood vessels in the homozygous animals as early as 14 days of postnatal age. The blood vessel deficiency at this early time coincides with degeneration of the photoreceptor cells and occurs at the approximate age when blood vessels in the normal mouse retina have reached maturity. After photoreceptor degeneration is complete, total blood vessel length per unit area continues to decrease from about one half of normal at the earlier ages to less than one third the amount at 1 yr and after.
Aging↗
Immunocytochemical localization of cellular retinol binding protein in the rat retina.
Cellular retinol binding protein (CRBP) was localized in the rat retina by means of light and electron microscopic immunocytochemistry. The peroxidase-antiperoxidase (PAP) method employed at the light microscopic level showed that CRBP is sharply localized to the retinal pigment epithelium (RPE). None was detectable in the epithelium of the pars plana or pars plicata of the ciliary body. Likewise, the photoreceptors were negative. Within the neural retina, the PAP method revealed a bilaminar staining pattern in the inner plexiform layer and staining within elements near the vitreal surface. Indirect immunoferritin electron microscopy demonstrated that CRBP is distributed uniformly within the RPE cytosol from basal infoldings to apical processes. The nucleoplasm also was stained, albeit, more lightly than the cytoplasm. Labeled elements in the inner plexiform layer and vitreal surface were identified as Müller cell processes and end feet, respectively. Interpretation of the results includes a dual role for CRBP in the RPE, namely its involvement in gene expression and transcytoplasmic transport of retinol.
Animals↗
Immunocytochemical localization of the main intrinsic polypeptide (MIP) in ultrathin frozen sections of rat lens.
The in situ distribution of the 26-kdalton Main Intrinsic Polypeptide (MIP or MP 26), a putative gap junction protein in ocular lens fibers, was defined at the electron microscope level using indirect immunoferritin labeling of ultrathin frozen sections of rat lens. MIP was found distributed throughout the plasma membrane of the lens fiber cell, with no apparent distinction between junctional and nonjunctional membrane. MIP was not detectable in the basal or lateral plasma membrane of the lens epithelial cell, including the interepithelial cell gap junctions; nor was MIP detectable in the plasma membrane or gap junctions of the hepatocyte. Previous reports have indicated that the protein composition of the lens fiber cell junction differs from that of the hepatocyte gap junction. The evidence presented here suggests that the composition of the fiber cell junction and plasma membrane is also immunocytochemically distinct from that of its progenitor, the lens epithelial cell.
Animals↗
Rhodopsin chromophore exchanges among opsin molecules in the dark.
Turnover of rhodopsin chromophore in vertebrate visual cells has been explored by light microscope autoradiography (LMARG) and radiobiochemical techniques. Retinol-binding protein (RBP) was isolated from human serum, its native ligand removed and replaced with [3H]-retinol. After reconstitution, [3H]-retinol-RBP was reassociated with prealbumin (PA), and the protein complex injected intravenously into dark-adapted animals. After selected intervals in the dark, animals were killed, and ocular tissues dissected under infrared illumination. Eyecups from frogs and mice were fixed (4 C) and after in situ reduction of the chromophore-protein linkage of rhodopsin with borane dimethyl amine (BDMA), processed histologically to retain lipids, or alternatively to extract them with chloroform-methanol (C-M), and LMARG performed. Rhodopsin was purified from detergent-solubilized mouse retinas by Concanavalin A (Con A) affinity chromatography and analyzed for radioactivity. Autoradiographic labeling of frog rod outer segments (ROS) was first detectable at 1 day postinjection, increasing over the duration of the experiment. At all times, label was distributed throughout the organelle in a diffuse pattern, although in certain cases a band of silver grains was also evident at the proximal end of the ROS, the site of new membrane assembly. Similar autoradiographic patterns were noted in mouse rods, although the kinetics of labeling differed in certain respects. In biochemical experiments, incorporation of [3H]-retinol into mouse rhodopsin was seen to occur very rapidly (less than 30 min), without an appreciable lag period. We interpret the diffuse labeling of ROS to result from an exchange in the dark of [3H]-vitamin A aldehyde for unlabeled opsin-bound chromophore, whereas the formation of a reaction band no doubt reflects the continual renewal of ROS membrane occurring in the dark. With respect to the former, the turnover of chromophore qualitatively resembles that found for membrane fatty acids.
Animals↗