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PubMed1956

Electron microscope observations on the submicroscopic organization of the retinal rods.

The submicroscopic organization of the retinal rods of the rabbit has been studied with high resolution electron microscopy in thin longitudinal and cross-sections. The outer rod segment consists of a stack of flattened sacs or cisternae each of them limited by a thin homogeneous membrane of about 30 A. The membrane of the rod sacs is attached to the surface membrane and is also in continuity with short tubular stalks of about 100 to 150 A which apparently end in relation with the connecting cilium. The bundle of filaments that constitute the connection between the outer and the inner segments is described under the name of connecting cilium. This fibrous component has a structure that is very similar to that of the cilium. It shows 9 pairs of peripheral filaments of about 160 A in diameter, a matrix material, and a surface membrane. Very infrequently two central single filaments are observed. The connecting cilium has a typical basal body in the inner segment; its distal end penetrates the outer segment, where it establishes some structural relation to the rod sacs. The relationships and submicroscopic organization of the connecting cilium were studied in longitudinal and in cross-sections passing at different levels of the rod segments. The inner rod segment shows two distinct regions: a distal and a proximal one. The distal region, corresponding to the ellipsoid of classical histology is mainly composed of longitudinally packed mitochondria. It also contains the basal body of the cilium, vacuoles of the endoplasmic reticulum, dense particles, and intervening matrix with very fine filaments. In the proximal region of the inner segment the mitochondria are lacking and within the matrix it is possible to recognize elements of the Golgi complex, vacuoles of the endoplasmic reticulum, dense particles and numerous neuroprotofibrils of 160 to 200 A in diameter which collect and form a definite bundle at the exit of the rod fiber. The interpretation of the connecting fibers as a portion of a cilium and of the outer segment as a differentiation of the distal part of a primitive cilium are discussed. The importance of the continuity of the surface membranes of the outer segment, connecting cilium, and inner segment is emphasized and its possible physiological role is discussed.

Animals↗
PubMed1983

Dependence of the single photon response on longitudinal position of absorption in toad rod outer segments.

Light responses were recorded from toad rods in order to study the dependence of the kinetics and amplitude of the single photon response on the longitudinal position of excitation within the rod outer segment. Membrane current was recorded from the inner segment of an isolated rod with a suction electrode while stimulating the outer segment with a dim transverse slit of light. Flashes at the tip of the outer segment gave smaller average responses than flashes at the base. Comparison of amplitude histograms from the two positions revealed that the fraction of incident photons eliciting an electrical response was the same at tip and base. Characteristic differences in flash sensitivity are therefore attributed to differences in the amplitude of the single photon response. Flash responses from the tip were slower than those from the base. For most cells, the tip response could be fitted by the same multistage filter equation that fitted the base response when only one of the filter time constants was increased. For both tip and base responses, the ensemble variance as a function of time was proportional to the square of the ensemble average. This indicates that single photon responses had the same wave form as their respective averages, and that there was no significant contribution of fluctuations in response latency to the wave form of the average. Background light reduced flash sensitivity at the tip more than at the base. The calculated cable attenuation of rod outer segments is not sufficient to explain the observed differences between the responses of the tip and base. The differences might instead be associated with a longitudinal gradient of internal sodium concentration, or with ageing of the outer segment discs. Calculations suggest that in the intact eye, the amplitude and time course of the average electrical response to absorption of a photon should depend slightly on the wave-length of the photon.

Animals↗
PubMed1989

Visualization of cyclic nucleotide binding sites in the vertebrate retina by fluorescence microscopy.

Cyclic nucleotides play a major role in cell signaling, especially in the nervous system. They act as cytoplasmic messengers in a wide range of physiological responses, but the spatial distribution of their sites of action within cells and tissues is not well-known. In the vertebrate retina, there is a class of well-characterized cGMP binding sites which control the permeability of cation channels in the rod outer segments (ROS), while cAMP is involved in several other systems in the inner retina. Biochemical studies of the cGMP-activated permeability in ROS have not distinguished between the subcellular compartments of disk and plasma membrane. By a new method using fluorescein-conjugated cyclic nucleotides, we have found strong cyclic GMP binding to the plasma membrane of the ROS, both on frozen sections of retina and in freshly isolated, leaky ROS. We also found a high density of cGMP binding sites on structures resembling the inner segment calycal processes. Little specific binding could be detected on the disk membranes or on any other retinal layer. In contrast, fluorescent cAMP did not label ROS, but gave a striking pattern of labeling on several deeper layers of the retina. These results suggest that the ROS plasma membrane has a much higher density of cGMP-controlled cation channels than the disk membranes, and point to other retinal layers where cAMP is likely to shape cellular responses. This method opens up novel morphological approaches to the study of cyclic nucleotide regulation.

Animals↗
PubMed1990

Effect of glycoconjugates on rod outer segment phagocytosis by retinal pigment epithelial explants in vitro assessed by a specific double radioimmunoassay procedure.

Rod outer segment (ROS) phagocytosis by explanted bovine retinal pigment epithelium (RPE) was evaluated by a procedure using an indirect double radioimmunoassay which distinguished between ROS attached to the RPE cell surface and those which had been ingested. This approach has been used to investigate the effect of a variety of glycoconjugates on the phagocytic process. Inclusion of the glycosaminoglycans (GAGs) chondroitin sulphate type-A (CS-A) and type-C (CS-C), hyaluronic acid (HA) or dermatan sulphate (DS) in the incubation medium significantly inhibited the ingestion phase of ROS phagocytosis, whereas the binding phase was inhibited to a lesser extent. The interphotoreceptor matrix (IPM), containing these GAGs as part of proteoglycans, also had an inhibitory effect on phagocytosis. The free monosaccharides mannose, fucose and galactose all stimulated the ingestion of ROS by RPE cells. These findings support the suggestion that glycoconjugates may have a physiological role in the photoreceptor renewal process.

Animals↗
PubMed1985

Interaction of hydrolysis-resistant analogs of cyclic GMP with the phosphodiesterase and light-sensitive channel of retinal rod outer segments.

cGMP opens cation-selective channels when applied to the cytoplasmic side of excised patches of membrane from retinal rod outer segments (ROS). If the light-sensitive channel in intact rods is gated only by cGMP, it should be possible to find a hydrolysis-resistant analog of cGMP that blocks the normal response to light by holding the channel open independent of the degree of illumination. We have studied the interaction of 8-bromo-cGMP (8-Br-cGMP) and the SP and RP phosphorothioate derivatives of cGMP [(Sp)-cGMP[S] and (RP)-cGMP[S]) with the cGMP phosphodiesterase (PDEase) of ROS, the cGMP-sensitive channel of excised ROS patches, and the light-sensitive channel of intact rods. All three analogs were hydrolyzed by PDEase much more slowly than was cGMP. The maximal rates of hydrolysis of 8-Br-cGMP, (SP)-cGMP[S], and (RP)-cGMP[S] were 7.3, 3.7, and less than 0.2 s-1, respectively, compared with 4000 s-1 for cGMP. These analogs are effective competitive inhibitors of the PDEase, with Ki values of 48, 25, and 90 microM, respectively. The nucleotide-activated conductances of excised patches were half-maximal at concentrations of 1.6, 210, and 1200 microM, respectively, compared with 17 microM for cGMP. Thus, 8-Br-cGMP is a highly potent channel agonist. The effects of these analogs on the dark current and photoresponses of intact rod cells were also measured. A suction electrode monitored membrane current across the ROS, while a patch electrode sealed on the inner segment was used to introduce a cGMP analog and to control membrane potential. All three analogs increased the dark current and markedly slowed the response to light flashes. 8-Br-cGMP increased the dark current of the outer segment as much as 48-fold. After the concentration of this analog had risen sufficiently, little of the current could be shut off by light, as expected of a direct effect on the light-sensitive channel of the plasma membrane. These results are consistent with the notions that (i) the light-sensitive channel of rods is controlled solely by the instantaneous concentration of cGMP and (ii) the cGMP-sensitive channel of excised patches is identical to the light-sensitive channel of intact rods.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1997

Crystallization and preliminary X-ray analysis of arrestin from bovine rod outer segment.

We present the first X-ray study of a member of the arrestin family, the bovine retinal arrestin. Arrestin is essential for the fine regulation and termination of the light-induced enzyme cascade in vertebrate rod outer segments. It plays an important role in quenching phototransduction by its ability to preferentially bind to phosphorylated light-activated rhodopsin. The crystals diffract between 3 angstroms and 3.5 angstroms (space group P2(1)2(1)2, cell dimensions a = 169.17 angstroms, b = 185.53 angstroms, c = 90.93 angstroms, T = 100 K). The asymmetric unit contains four molecules with a solvent content of 68.5% by volume.

Animals↗
PubMed1980

Light-enhanced cross-linking of rhodopsin in rod outer segment membranes as detected by chemical probes.

Bovine rod outer segment membranes were treated with cross-linking reagents before and after light exposure. Bleached membranes showed enhanced cross-linking with difluorodinitrobenzene or methyl acetimidate compared to dark-adapted membranes. The light-induced enhancement of cross-linking may be due to increased association of rhodopsin monomers in the light and/or due to increased reactivity of amino and sulfhydryl groups of bleached rhodopsin. In some instances, the band ascribed to the rhodopsin monomer in gel electrophoresis appears as a partially resolved doublet. Treatment of bleached rod outer segment membranes with methyl acetimidate improved the resolution of the doublet into two closely migrating bands.

Animals↗
PubMed1984

Immunologic characterization of the photoreceptor outer segment cyclic GMP phosphodiesterase.

High affinity (KD approximately 1 X 10(-9) M) monoclonal antibodies (ROS-1 and ROS-2) were prepared to bovine photoreceptor outer segment cGMP phosphodiesterase. ROS-1 immunoadsorbed greater than 95% of the cGMP phosphodiesterase activity from a detergent-solubilized bovine retina extract while ROS-2 immunoadsorbed only a subfraction of the same activity. Sodium dodecyl sulfate gel analysis of these immunoadsorbates demonstrated that ROS-1 and ROS-2 specifically adsorbed only peptides that comigrated with purified rod outer segment phosphodiesterase. Limited trypsin digestion of purified rod outer segment phosphodiesterase greatly reduced its affinity for ROS-1 but not ROS-2. When a crude heat-stable inhibitor fraction was added back to the activated enzyme, the affinity for ROS-1 was restored, suggesting that the inhibitor was necessary for ROS-1 binding. ROS-1 but not ROS-2 was found to inhibit cGMP phosphodiesterase which had been activated either by dilution or guanyl nucleotide. The inhibitory property of ROS-1 may provide a useful probe for directly studying the effects of this phosphodiesterase on the phototransduction response in the retina. Sodium dodecyl sulfate gel analysis demonstrated that the ROS-1 immunoadsorbates from mammals, fish, and amphibia contained peptides of similar mobility. Immunocytochemistry performed with ROS-1 and fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG localized the antigenic determinant to both rod and cone outer segments suggesting the presence of an antigenically similar phosphodiesterase in both types of photoreceptors.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

Fatty acid composition of phospholipids from chick neural retina during development.

The fatty acid composition of retina phospholipids from developing chicks was investigated to determine what changes, if any, occur in the relative levels of polyunsaturated fatty acids. Embryonic chicks were killed at 3-day intervals from day 6 through hatching (day 21), and at 1 week post-hatch. Fatty acids were prepared from retina phospholipids and were analysed by capillary gas-liquid chromatography. A comparison of the composition of yolk taken on day 6 with retinas isolated on that day revealed a much greater proportion of polyunsaturated fatty acids in the latter, suggesting an ability of the embryo to metabolize selectively unsaturated fatty acids at this early stage of development. Throughout the time course studied, saturated fatty acids constituted 50% of all fatty acids, most of which was due to palmitic acid (16:0; 33-41%). Among other saturated fatty acids, myristic acid (14:0) increased to maximal levels by day 18, then declined, while stearic acid (18:0) was minimal on day 12 and then increased. Polyunsaturated fatty acids varied between 14 and 23% of total fatty acids, depending on the developmental stage. One of the most remarkable changes in polyunsaturated fatty acids occurred in the levels of 22:4 (n-6). The proportion of this single fatty acid decreased from 9.4 to 2.4% between days 15 and 18. Relative levels of 22:5 (n-6) increased significantly between day 21 and 1 week post-hatch, from 1.1 to 3.2%. In this same time period, the proportion of 22:6 (n-3), the fatty acid known to be prominent in the outer segments of rod-dominant retinas, did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Kinetics of light-dependent Ca fluxes across the plasma membrane of rod outer segments. A dynamic model of the regulation of the cytoplasmic Ca concentration.

We measured simultaneously in single toad rods the membrane photocurrent and the Ca concentration in a small volume surrounding the outer segment. Illumination causes a rise in the extracellular Ca concentration. Photocurrents and Ca concentration changes occur over the same range of light intensities. Analysis of the time course of the Ca concentration changes suggests that these concentration changes arise from the difference in the transport rates of light-activated Ca influx and efflux across the outer segment plasma membrane. The Ca influx occurs through the light-sensitive channels of the outer segment membrane and the efflux through Na/Ca exchangers. In 0.1 mM external Ca, approximately 1-2% of the dark current is carried by Ca ions. The Ca efflux in the dark is identical to the influx, approximately 2 X 10(6) ions/s. Upon illumination, the Ca influx decreases with a time course and light sensitivity identical to those of the photocurrent. The Ca efflux, on the other hand, has very different kinetics from those of the photocurrent. Upon illumination, the Ca efflux decreases with a time course and light sensitivity determined by the change in membrane voltage and in the free cytoplasmic Ca concentration near the plasma membrane. In response to bright stimuli, which saturate the photocurrent for prolonged periods of time, the Ca efflux decays with an exponential time course from its value in darkness. The average time constant of this decay is 2.5 s. From the kinetics of the light-activated Ca fluxes, it is possible to predict that illumination causes a decrease in the cytoplasmic Ca concentration. We present a model of the regulation of the cytoplasmic Ca concentration by the dynamic balance of the Ca influx and efflux from the rod outer segment. The model accounts for our experimental observations and allows us to predict the time course and extent of the light-dependent decrease in the free cytoplasmic concentration.

Animals↗
PubMed1994

Inositol triphosphate generation in cultured rat retinal pigment epithelium.

PURPOSE: To evaluate the generation of the second messenger molecule inositol triphosphate (IP3) in response to phagocytic challenge in cultures of retinal pigment epithelium (RPE) prepared from normal Long Evans or dystrophic Royal College of Surgeons (RCS) rats. METHODS: RPE cultures were phagocytically challenged with isolated rat rod outer segments (ROS) or polystyrene latex spheres (PSL). Carbachol was added to some cultures as a positive control to stimulate the IP3 pathway. Inositol triphosphate levels in RPE cells were analyzed by high-pressure liquid chromatography and radioreceptor assay. Incorporation of 3H-myoinositol into phosphatidylinositol lipids was analyzed by thin layer chromatography and autoradiography. RESULTS: Long Evans RPE phagocytically challenged with ROS or exposed to carbachol showed a significant increase in IP3 levels compared to unchallenged control cultures or cultures phagocytically challenged with PSL. RCS RPE cells did not show an increase in IP3 levels upon phagocytic challenge with ROS or PSL. Carbachol treatment of RCS RPE produced an increase in IP3 levels, demonstrating that the components of the IP3 pathway are present and the pathway can be activated. Phagocytic challenge of Long Evans RPE with ROS was associated with a decrease in the IP3 precursor, phosphatidylinositol bisphosphate (PIP2). No decrease in PIP2 was observed in RCS RPE incubated with ROS. CONCLUSIONS: These results show that phagocytic challenge of Long Evans RPE with ROS is associated with PIP2 hydrolysis and subsequent generation of IP3 as a second messenger. In RCS RPE, ROS stimulation of the IP3 pathway is absent.

Animals↗
PubMed1992

Zymosan induced 45Ca uptake by retinal pigment epithelial cells.

45Ca uptake was studied in isolated frog retinal pigment epithelial cells in response to the phagocytic stimuli, zymosan. 45Ca uptake was strongly stimulated immediately in the presence of zymosan particles. Calcium uptake was proportional to the zymosan concentrations. After 60 min in the presence of zymosan acid phosphatase and beta-glucuronidase activities showed a 25% and 50% increase, respectively. Rod outer segments induced a similar increase of these enzyme activities. The zymosan-induced lysosomal enzyme activities was inhibited by cytochalasin B and ruthenium red. The ionophore A23187 produced a remarkable increase in 45Ca uptake but did not affect the lysosomal enzyme activities. These results suggest that in vitro RPE cells are able to respond to zymosan as phagocytosable stimuli and that calcium mediate that response.

Acid Phosphatase↗
PubMed1996

Functional characterization of a guanylyl cyclase-activating protein from vertebrate rods. Cloning, heterologous expression, and localization.

The membrane-bound guanylyl cyclase in vertebrate photoreceptor cells is one of the key enzymes in visual transduction. It is highly sensitive to the free calcium concentration ([Ca2+]). The activation process is cooperative and mediated by a novel calcium-binding protein named GCAP (guanylyl cyclase-activating protein). We isolated GCAP from bovine rod outer segments, determined amino acid sequences of proteolytically obtained peptides, and cloned its gene. The Ca2+-bound form of native GCAP has an apparent molecular mass of 20.5 kDa and the Ca2+-free form of 25 kDa as determined by SDS-polyacrylamide gel electrophoresis. Recombinant GCAP was functionally expressed in Escherichia coli. Activation of guanylyl cyclase in vertebrate photoreceptor cells by native acylated GCAP was half-maximal at 100 nM free [Ca2+] with a Hill coefficient of 2.5. Activation by recombinant nonacylated GCAP showed a lower degree of cooperativity (n = 2.0), and half-maximal activation was shifted to 261 nM free [Ca2+]. Immunocytochemically we localized GCAP only in rod and cone cells of a bovine retina.

Amino Acid Sequence↗
PubMed1988

Phospholipase C activity and substrate specificity in frog photoreceptors.

The photoreceptor, like many other cells, undergoes receptor-mediated breakdown of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. The lack of phosphatidylinositol (PdtIns) breakdown during receptor stimulation suggests the existence of a phospholipase C specific for polyphosphoinositides. Phospholipase C activity in frog rod outer segments was assayed with several substrates. The activity was selective for the polyphosphoinositides, phosphatidylinositol 4-phosphate [PtdIns(4)P] and PtdIns(4,5)P2. PtdIns was hydrolysed at 2% of the rate of hydrolysis of PtdIns(4,5)P2. No activity was detected when phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine were used as substrates. The enzymatic activity was optimal at neutral pH. These findings suggest, but do not prove, that this activity might contain the light-regulated phospholipase C of the photoreceptor.

Animals↗
PubMed1991

Light damage in the rat retina: the effect of dietary deprivation of N-3 fatty acids on acute structural alterations.

We investigated the effect of depleting membrane docosahexaenoic acid (DHA, 22:6n-3) content through dietary deprivation of n-3 fatty acids on the susceptibility of the photoreceptors and pigment epithelium cells to acute light-induced changes. Male Sprague-Dawley rats were raised throughout gestation, lactation and up to the age of 8 weeks on semi-purified diets containing either safflower oil (SFO, n-3 deficient diet) or soybean oil (SO) as the sole source of lipids. A third group was switched at weaning from safflower oil to soybean oil (SFO/SO). Rats were maintained on a 12 hr/12 hr light/dark cycle in which the light level at the front of the cages was 5-10 lx. Light damage was produced by exposing dark-adapted animals to diffuse white fluorescent light of 700-800 lx for 30 min followed by 90 min of darkness. In order to study recovery from light damage, additional groups of SFO and SO rats were returned to dim cyclic light for 27 hr following bright light exposure. DHA content in retinal phosphatidylethanolamine and phosphatidylcholine was 65-75% lower in rats fed SFO than in rats fed SO. The decrease was compensated for by an increase in 22:5n-6, the total content of polyunsaturated fatty acids (PUFA) being similar in both the SFO and SO groups. The SFO/SO rats had DHA levels similar to SO animals, but 22:5n-6 remained elevated resulting in a slightly higher level of total PUFA. Severe rod outer segment (ROS) membrane disruptions were seen following bright light exposure in rats on the SO and SFO/SO diets. The appearance of these disruptions did not change significantly during more than 24 hr in dim cyclic light. In contrast, there were virtually no acute ROS lesions in the SFO group. Furthermore, there was a strong light-elicited disk-shedding response in the SO rats but not in the other two groups. The pigment epithelium of the DHA deficient retinas showed a significantly greater accumulation of large lipid droplets in the dark-adapted state. Notably, whole retina rhodopsin levels were 15% higher in the SFO than in the SO group. These results indicate that depletion of retinal DHA reduces the susceptibility of the rod outer segments to acute light damage and at the same time may alter visual pigment photochemistry and other photoreceptor and pigment epithelium functions.

Animals↗
PubMed1995

Ultrastructural features of retinal dystrophy in mutant vitiligo mice.

Ultrastructural features of the retinal pigment epithelium and photoreceptor cells were studied in vitiligo (C57BL/6 mivit/mivit) mice. Eyes from 12-day- to 56-week-old animals were analysed. Abnormal photoreceptors were seen in 12-day-old mice. By 3 weeks malformed outer segments were evident in the posterior and equatorial retina, but normal photoreceptors were present in the periphery. By 28 weeks, a marked gradient in cell loss was evident, with a progressive increase in cell viability along the posterior-peripheral axis. Viable intact photoreceptors were still present in the peripheral retina of 56-week-old mice. Melanosome content varied between adjacent pigment epithelium cells in both the posterior and peripheral retina. In the choroid, however, a steep posterior-peripheral gradient in melanosome content was evident with highest pigmentation in the periphery. In the optic nerve head region abnormal development of photoreceptors was correlated with proliferation of abnormal pigment epithelium cells. Accumulation of rod outer segment debris in the posterior and peripheral subretinal space preceded photoreceptor cell death. Short pigment epithelial microvilli without proper attachment to photoreceptors are suggestive of alterations in pigment-epithelium-photoreceptor interaction, which might affect photoreceptor differentiation and phagocytosis of rod outer segments by pigment epithelium cells.

Animals↗