Outer Segments Of Photoreceptor Cells Research Articles

The relative proportions of lysosomal enzyme activities in bovine retinal pigment epithelium

The lysosome fractions from bovine retina, liver and retinal pigment epithelium were isolated by subcellular fractionation and compared with regard to the relative proportions of several hydrolytic enzyme activities. It was found that the lysosome fraction of the retinal pigment epithelium is more than three times as active as the lysosome fractions from other tissues in degrading the rhodopsin of photoreceptor (rod) cell outer segments. This proteolytic activity is attributable to a cathepsin D-like proteinase, and the possible biochemical bases for its increased activity in the pigment epithelium are discussed, including interaction with phospholipase A. It is suggested that the lysosomes of the retinal pigment epithelium are specialized in their content of hydrolytic enzymes for the degradation of photoreceptor cell outer segments.

22] Preparation and properties of dispersed rat retinal cells

This chapter presents a rapid method to disperse rat retinas enzymically and separate large quantities of various cell types by centrifugation through a Metrizamide density gradient. This method has been useful for biochemical and pharmacological characterization of several types of retinal cells. Using phase-contrast microscopy, the neurons are primarily rounded and could not be identified without further biochemical characterization photoreceptor cell outer rod segments and may be used as a biochemical marker for these cells. Data describing the biochemical characterization of the whole retina are difficult to interpret because of the heterogeneity of cell types. Consequently, the isolation of enriched populations of purified retinal cells represents a breakthrough in the studies of retinal cell function. The technique for isolation of retinal cells involves chemical dispersion of whole retina using collagenase followed by nonequilibrium centrifugation through a Metrizamide continuous density gradient. This technique has several advantages: large quantities of cells are recovered, enabling thorough biochemical characterization of the cells, and the dispersion is rapid (the cells are exposed to collagenase for 12 min).

9] Preparation and characterization of sealed bovine rod cell outer segments

Publisher Summary This chapter discusses the preparation and characterization of sealed bovine rod cell outer segments. Mammalian rod (photoreceptor) cell outer segments consist of closely packed membrane disks surrounded by the plasma membrane. They can easily be purified because they are large, have a very low buoyant density (resulting from a high lipid content), and detach easily from the parent photoreceptor cell. Earlier procedures for isolating rod outer segments (ROS) yielded preparations with a low or variable content of these proteins, probably because most of the plasma membranes were extensively disrupted or because the discontinuous gradients that are usually employed did not separate sealed from disrupted ROS. The sealed ROS can be separated from disrupted ROS and disk membranes by use of continuous isopycnic density gradients, a method that takes advantage of the differences in contents and osmotic properties of the two kinds of particles. ROS are identified by their high rhodopsin content. The characteristic density of sealed ROS, their clean separation from disrupted ROS, and the characteristic ratios of protein to rhodopsin in the two fractions are usually sufficient for the identification of the sealed fraction; however, the following other methods may be useful.

Hexachlorophene retinopathy in suckling rats: A light- and electron-microscopic study of short-term and long-term effect of hexachlorophene intoxication on the retina of young rats

Hexachlorophene (HCP) in very large doses has a toxic effect on the retina of adult rats. The present study examined the sensitivity of the developing retina to smaller doses and its ability to recover from neurotoxic damage. Suckling rats were intoxicated with HCP added to the mothers' diet. The retinae of the pups were examined by light and electron microscopy for early and late effects. All dose levels above 100 ppm caused vacuolar degeneration of the outer segments of photoreceptor cells within 14 days of intoxication, followed by degeneration of the inner segments after 28 days. The cell bodies of photoreceptor cells and all other retinal layers remained normal. Sucklings intoxicated with HCP (100–500 ppm) added to the mothers' diet for 28 days and then withdrawn, showed no recovery of the outer or inner segments of photoreceptors when examined up to 150 days later. In addition, extensive degeneration and loss of photoreceptor cell bodies occurred after withdrawal of HCP and the degenerative process extended to involve the inner nuclear layer. The rate of appearance of pathological changes was proportional to the total amount of ingested HCP. This study showed that photoreceptor cells in newborn rats are highly sensitive to HCP. The retinal lesions were irreversible and continued to progress by continued trans-synaptic degeneration after withdrawal of the toxic agent.

Immunocytochemical localization of the D2-protein in rat retina

The localization of the D2-protein in rat retina was investigated by quantitative immunoelectrophoresis and by immunoperoxidase staining on both the light microscopic and the electron microscopic level. The D2-protein was enriched 1.4 times in retina compared to whole brain and it was located to most presynaptic and some postsynaptic membranes in the inner and outer plexiform layers. Moreover, the D2-protein was located on the cell membrane confining the ciliary neck region connecting the inner and the outer segments of photoreceptor cells. Thus, on the electron microscopic level, the D2-protein is not restricted to the presynaptic membrane, but it is also found in other parts of the neuronal surface membrane.

Histogenesis of the ferret retina

The development of the ferret retina was studied with light and transmission electron microscopy from prenatal day 21 (E21; midgestation), through postnatal day 33 (P33; eye opening). Tissue samples were taken from the posterior pole of the retina. At E21, the optic cup was completely formed, the pigment epithelium was a single cell layer and the sensory retina consisted of an inner marginal layer and an outer layer of neuralblastic cell perikarya. Mitotic figures were observed among the nuclei of the outermost layer of neuralblastic cells through P10. At E27, the layer of neurablastic cell perikarya was divided into inner (light-staining) and outer (dark-staining) layers. The inner layer, presumbbly ganglion cells, was separated from the outer dark-staining layer at E36; at, E39, a distinct ganglion cell layer and inner plexiform layer was present. At birth light-staining nuclei each with a central dark-staining nucleolus were observed among the nuclei of the outermost layer of neuralblastic cells; these nuclei were presumed to be developing cone cell nuclei. At P2 light-staining nuclei, presumably horizontal cells, appeared within the remaining layer of neuralblastic cell perikarya. Conventional synapses were seen in the inner plexiform layer on P4. Separation of the layer of neuralblastic cell bodies into inner and outer nuclear layers was evident, on P9; by P12, the layers were clearly separated by the outer plexiform layer. Synaptic ribbons appeared in the outer plexiform layer on P12 and in the inner plexiform layer by P15. At the beginning of the second postnatal week (P15) membranous disks were seen in some photoreceptor cell outer segments. Vasculature was not observed prenatally; however, by the fifth week postnatally capillaries were observed as deep as the inner nuclear layer. From this study it is evident that, the ferret retina provides an easily obtainable postnatal tissue at an early stage of development.

Isolation procedure for bovine retinal rod outer segments.

An isolation procedure to obtain rod outer segments from cattle retinas is reported. Centrifugation of homogenates in discontinuous and continuous sucrose density gradients yields purified photoreceptor cell outer segments. Assay of the final preparation for rhodopsin content gives a ratio of 2.4 for DO280nm/DO498nm.

Stimulatory effect of taurine on Ca-uptake by disc membranes from photoreceptor cell outer segments

Effects of taurine and related compounds on Ca-uptake by the disc membranes prepared from dark-adapted frog retina were studied. Taurine stimulated ATP-dependent Ca-uptake and the turnover of 45Ca in the disc membranes without affecting basal activity, but it was not observed with the synaptic plasma membranes from rat brain. The stimulatory effect appears to be specific to taurine, since cysteine sulfinic acid, hypotaurine, isethionic acid, β-alanine and γ-aminobutyric acid (GABA) did not stimulate Ca-uptake. The maximal activation, observed at about 30 mM taurine, was about 3 fold, and the Km value for taurine was 10 mM. These results might suggest that taurine modifies translocation of Ca ion in the rod outer segment.

Histogenesis of the cat retina

The development of the retina of the cat was studied with light and transmission electron microscopy from prenatal day 36 (E36) through postnatal day 9 (P9; eye opening), and at the adult stage (1 year). Tissue samples were taken from the posterior pole of the retina. At E36, the optic cup was completely formed, the pigment epithelium was a single cell layer, the retina consisted of an inner marginal fiber layer and an outer layer of neuroblastic cells, and the innermost cells of the neuroblastic cell layer, presumptive ganglion cells (which might still migrate through the inner plexiform layer) were displacing away from the neuroblastic cell mass. At E40, a distinct ganglion cell layer was seen. At E46, primitive horizontal cells appeared within the neuroblastic cell layer. Separation of the neuroblastic cell mass into inner and outer nuclear layers was first evident on E48; by E60, the layers were clearly separate. Conventional synapses were seen in the inner plexiform layer in the week prior to birth. Blood vasculature was observed prenatally as deep as the inner plexiform layer. In the newborn a few discs were seen in some photoreceptor cell outer segments. Synaptic ribbons appeared first in the outer plexiform layer in the newborn and then in the inner plexiform layer by P4.

Freeze-fracture analysis of photoreceptor cell outer segment disks after minimal extraction of rhodopsin

The outer segment disk membranes in the photoreceptor cells of the guinea pig retinas were analyzed after partial extraction of rhodopsin with cetyltrimethylammonium bromide (CTAB). During the earliest stages of rhodopsin extraction, the particulate structure of the particulate face of the disk membranes becomes less dense with the appearance of smooth areas extending between the particles. These smooth areas could occupy a large part of the particulate membrane face. The disk membranes could still retain a planar structure. A transformation of the disk membranes into small vesicles was also evident and was considered to correspond to a stage where the extraction has progressed beyond the earliest stages. Extraction with digitonin led to an immediate disintegration of the disk membranes into vesicles. Since CTAB can preferentially solubilize rhodopsin without extracting the membrane lipids (Heller, 1968;Biochemistry, 7, 2906), the observations were considered to support the interpretation of Sjöstrand and Kreman (1978,J. Ultrastruct. Res., 65, 195), that the surface structure of the particulate membrane face is due predominantly to rhodopsin molecules forming a separate layer in the disk membrane.

Membrane-dependent guanine nucleotide binding and GTPase activities of soluble protein from bovine rod cell outer segments.

Soluble proteins can be extracted by osmotic shock of purified rod (photoreceptor cell) outer segments that have intact plasma membranes. The soluble proteins include a component that contains tightly bound GDP-Exchange of this GDP with exogenous nucleotide is catalyzed by (and requires) the membranes from the outer segments. ATP does not participate in these reactions. Approximately one-half of the binding sites in the soluble component require GTP as the source of exogenous nucleotide; the remainder accept GTP or GDP with equal facility. When exogenous GTP is the source of bound nucleotide, it is found in the complex in the form of GDP. Exchange of bound nucleotide with GTP is stoichiometrically related to GTPase activity; this activity is highly dependent upon the presence of both membranes and soluble protein. The soluble nucleotide binding protein was purified by making use of the fact that it binds tightly to the membranes (under conditions of moderate ionic strength) in the absence of GTP and can be eluted by solutions containing low concentrations of GTP (but not GDP or ATP, nor can it be eluted by GTP-free solutions of low ionic strength). The purified protein contains two polypeptide chains of molecular weights 41,000 and 37,000; these are the major species that can be extracted from the outer segments by osmotic shock, and they constitute approximately 7% of the total protein of the isolated organelle.

Freeze-fracture analysis of structure of plasma membrane of photoreceptor cell outer segments

The plasma membrane of the outer segments of photoreceptor cells in the guinea pig retina was analyzed by means of freeze-fracturing. Two fracture faces with greatly different structure were observed. The relationship of these two faces to the edge which was exposed when the fracture passed across the membrane made it possible to establish that they revealed the cytoplasmic and the peripheral surfaces of the plasma membrane. The thickness of the plasma membrane was measured to be about 100 A. The difference in appearance of the cytoplasmic and the peripheral surfaces of the plasma membrane was interpreted to reveal a layered structure with a predominantly lipid layer at the cytoplasmic surface, and a predominantly protein layer at the peripheral surface. A concept describing the molecular structure of the membrane is proposed. The outer segment plasma membrane differs structurally from the outer segment disk membranes. Certain aspects of disk membrane formation as invaginations of the plasma membrane are discussed. The fact that only two fracture faces were observed and no complementary faces was explained by a simple analysis of some effects of the fracturing on the frozen material.

Molecular structure of outer segment disks in photoreceptor cells

The structure of the photoreceptor cell outer segment disks in guinea pig, frog, and gecko retinas were analyzed by means of freeze-fracturing and showed basically identical features. Thanks to a rather accurate locating of the fracture planes based on observations, the observed structure could be interpreted to reveal that the outer segment disk membranes consist of two layers with greatly different physical properties and with each layer measuring about 50 A in thickness. These observations were interpreted on the basis of information obtained earlier by means of polarization optical and x-ray diffraction analysis as well as of what is known about the chemical composition of the photoreceptor cell outer segments. On this basis it was concluded that each disk membrane consists of one lipid bilayer and one monolayer of photopigment molecules dispersed in an aqueous medium of small volume. A particulate structure at the edge of the disk made this part of the disk appear as structurally different from the disk membrane. A hypothetical mechanism is proposed for the transmission of a signal linking energy transduction to the change in permeability of the outer segment plasma membrane caused by absorption of light. The outer segment disk membrane differs structurally extensively from the inner mitochondrial membranes, and the differences were interpreted to reflect the very different functions at a molecular level of these two types of membranes.

Evidence for Myosin Associated with Bovine Photoreceptor Cell Outer Segments

Conference Article| December 01 1978 Evidence for Myosin Associated with Bovine Photoreceptor Cell Outer Segments JOHN HESKETH; JOHN HESKETH * Search for other works by this author on: This Site PubMed Google Scholar NOELLE VIRMAUX; NOELLE VIRMAUX 1Centre de Neurochimie du CNRS, 11 rue Humann, 67085 Strasbourg Cedex, France Search for other works by this author on: This Site PubMed Google Scholar DOMINIQUE AUNIS DOMINIQUE AUNIS 1Centre de Neurochimie du CNRS, 11 rue Humann, 67085 Strasbourg Cedex, France Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1978) 6 (6): 1271–1272. https://doi.org/10.1042/bst0061271 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation JOHN HESKETH, NOELLE VIRMAUX, DOMINIQUE AUNIS; Evidence for Myosin Associated with Bovine Photoreceptor Cell Outer Segments. Biochem Soc Trans 1 December 1978; 6 (6): 1271–1272. doi: https://doi.org/10.1042/bst0061271 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1978 Biochemical Society1978 Article PDF first page preview Close Modal You do not currently have access to this content.

Distribution of enzyme activities in subcellular fractions of bovine retina.

Centrifugation of homogenates of bovine retinas to isopycnic equilibrium in sucrose density gradients yielded three partially overlapping bands of particles which were, in the order of increasing density: (a) photoreceptor cell (rod) outer segments; (b) plasma membranes, lysosomes, and large fragments of endoplasmic reticulum; and (c) mitochondria. The only enzyme activity investigated which had a peak coinciding only with outer segment fractions was guanylate cyclase. Enzyme activities with peaks in both the outer segment and denser fractions included 5'-nucleotidase and cyclic GMP phosphodiesterase. Enzyme activities with peaks only in the denser fractions included sodium and potassium ion-activated ATPase ((Na+ + K+)-ATPase), adenylate cyclase, cyclic AMP phosphodiesterase, beta-glucosidase, beta-galactosidase, and succinate-dependent cytochrome c reductase. These results suggest that some of the activities once thought to be present in rod outer segments are actually present in particles from elsewhere in the retina which contaminate rod outer segment preparations.

Light and electron microscopic radioautography of glucose-6-H 3 incorporation in the retina of the adult newt, Triturus viridescens dorsalis.

AbstractTritiated glucose was administered to fasted newts to study glucose incorporation in the retina and particularly the photoreceptor cells. Incorporation of glucose into glycogen throughout the retina commenced at 30 minutes after injection. The first silver grain concentrations were observed over the photoreceptor myoid at two hours. Incorporation of glucose into glycogen reached a maximum at six hours with grains concentrated over photoreceptor paraboloids, the apices of Müller cells and the inner and outer plexiform layers. By 24 hours there was a reduction of the label in glycogen but silver grains were now visible over mitochondria in the photoreceptor ellipsoids and in increased amounts in the outer and inner plexiform layers and the nerve fiber layer. After three weeks, most radioactive material was observed over the photoreceptor cell outer segments. These results suggest that glycogen is added at the inner aspect of the photoreceptor paraboloid and then moves outward in a scleral direction, where it is metabolized by the ellipsoidal mitochondria.

A lipid histochemical study of the rat retina damaged by visible light

Adult male rats were exposed to intense light at a body temperature of about 39°C for 2 half-hour periods with an interval of 2 hr in the dark. Under these standardized conditions, this treatment caused a highly reproducible degeneration of all the epithelial cells and all the visual cells. The histochemically demonstrable changes in the lipid pattern were examined at different time intervals. The outer segments of the visual cells in the normal rat retina are characterized by a strong reaction to phospholipids and a moderate reaction with the modified PAS method. They show less intense reactions with other histochemical lipid methods. The light-induced degeneration of the epithelial and rod cells occurs in three distinct stages. During the first phase, lasting about 2 days, there is rapidly progressive structural disintegration and edema in the scleral half of the retina, without any significant change in the lipid pattern. During the second phase, characterized by phagocytosis and removal of debris from the retina, there are no qualitative changes in the histochemical lipid pattern, although the intensities of the lipid reactions rapidly diminish. Most of the lipid material is removed during the first days of the second stage. Almost no reacting lipids are present during the third reparative stage. Eventually, the retina consists only of the five vitreous layers separated from the choroid by a single cell layer. There is good correlation between the histochemical and biochemical lipid patterns of the outer segments of the photoreceptor cells. The changes in the histochemical lipid pattern in the light-damaged retina are different from those occurring during Wallerian degeneration in spite of the ultrastructural similarities between the outer segments of photoreceptor cells and the myelin. Debris is removed by phagocytizing cells in the same manner in both of these damaged tissues, but the process is much more rapid in the light-damaged retina.

Visual pigments of frog and tadpole ( Rana pipiens)

Photoreceptor cell outer segments of five different morphologies from frog and tadpole retinas have been examined by microspectrophotometry. Photosensitive pigments were found with the following λ max : Frog: red rod 502, green rod 432, principal cone 575, accessory cone 502, single cone 575; Tadpole: red rod 527, green rod 438, principal cone 620, accessory cone 527, and single cone 620. Axial densities of pigments are computed from their measured transverse densities. A 2 derived pigments have wider bandwidths on a wavenumber plot than A 1 based pigments. Red rod wavenumber plots match published nomograms but other pigments do not. All pigments are dichroic and have comparable photosensitivities. Molar concentration of visual pigments is nearly the same in all cells. However, the small size and number of cones in frog retinas probably makes the total relative contribution of cone pigment molecules too small for successful spectroscopic studies in extracts. Animals in the visual pigment phase of metamorphosis have identical pigment mixtures in all red rods.

Sequence of Changes in the Fine Structure of the Retina of Dogs Irradiated During Retinal Maturation

It has been known since early in this century that exposure to ionizing radiation during maturation of the mammalian retina results in necrosis of post-mitotic differentiating cells in the nuclear layer, followed by marked retinal dysplasia with rosette formation, and partial to complete agenesis of outer segments of photoreceptor cells. The effect has been morphologically related to the area of the retina undergoing differentiation at the time of irradiation. Alteration in spacial and pressure relationships are said to result in the tubular rosette formations. These effects have been described in mice, rats, monkeys, dogs and man. Doses less than 25 R have produced severe dysplasia in the developing retina of mice. The mature retina is damaged only by doses of several hundred to a few thousand R.

The renewal of photoreceptor cell outer segments.

The utilization of methionine-(3)H by retinal photoreceptor cells has been studied by radioautographic technique in the rat, mouse, and frog. In all three species, the labeled amino acid is concentrated initially in the inner segment of the cell. Within 24 hr, the radioactive material is displaced to the base of the outer segment, where it accumulates as a distinct reaction band. The reaction band then gradually moves along the outer segment and ultimately disappears at the apex of the cell, which is in contact with the retinal pigment epithelium. These findings are interpreted to indicate that the photoreceptor cell outer segment is continually renewed, by the repeated lamellar apposition of material (membranous discs) at the base of the outer segment, in conjunction with a balanced removal of material at its apex. The outer segment renewal rate is accelerated in frogs when ambient temperature is raised, and is elevated in both frogs and rats when the intensity of retinal illumination is increased.