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PubMedDate not supplied

Developmental expression of the rat rod photoreceptor cGMP-gated cation channel.

The appearance of cGMP-gated cation channel protein in the postnatal rat retina has been studied by fluorescence immunocytochemistry of radial retinal sections and immunoblots of retinal membrane proteins. Channel immunoreactivity was first detectable with RCNGC1-7H2 monoclonal antibody at postnatal day 7 (PN7) by both methods. Immunocytochemical label in retinal sections was localized to the outer segments, and immunoreactivity increased with increasing age. We also compared the developmental appearance of the cGMP-gated cation channel to that of other phototransduction proteins and developmental markers. RET-P2, a monoclonal antibody recognizing the 39-kDa rds/peripherin disc protein, first labeled outer segments at PN7, coincident with cGMP-gated cation channel expression. Double labeling of the same section of PN7 rat retina with RET-P2 and R309 (a polyclonal antiserum against the rod cGMP-gated cation channel) revealed identical patterns of labelling. Similarly, double labeling with RCNGC1-7H2 and an antibody against the rod cGMP-phosphodiesterase gave coincident labeling, suggesting coordinate expression mechanisms of phototransduction proteins with each other and with outer segment structural proteins.

Animals↗
PubMed1984

Actin in the photoreceptor connecting cilium: immunocytochemical localization to the site of outer segment disk formation.

Actin has been localized in Rana pipiens retinas that were fixed and embedded in aldehyde cross-linked BSA. Thin sections were reacted sequentially with (a) affinity-purified antiactin antibodies induced in rabbits; (b) biotinyl-sheep anti-rabbit antibodies; and (c) avidin-ferritin conjugates. As expected, antiactin labeling density was high in the apical pigment epithelial cell processes and in the calycal processes of photoreceptors. Actin was also localized in a new site. The connecting cilium that joins the inner and outer segments of both rods and cones was heavily labeled by antiactin at its outer segment (OS), or distal, end. In this region of the cilium, the plasma membrane evaginates to form new OS disks and these basal disks were labeled in some instances. Below the new disks in rods, the cytoplasm of liplike expansions of the distal cilium was also heavily labeled. The plasma membrane and interior of the connecting cilium and the remainder of the OS were unlabeled. These findings suggest that actin may participate in the vectorial transport of opsin and other intrinsic membrane proteins that are incorporated into newly forming OS disks. The results also implicate actin in the membrane expansion involved with OS disk formation.

Actins↗
PubMedDate not supplied

Light-induced reduction of cytoplasmic free calcium in retinal rod outer segment.

The response of retinal rod photoreceptors to light consists of a membrane hyperpolarization resulting from the decrease of a light-sensitive conductance in the outer segment. According to the calcium hypothesis, this conductance is blocked by a rise in intracellular free Ca triggered by light, a notion supported by the findings that an induced rise in internal Ca leads to blockage of the light-sensitive conductance and that light triggers a net Ca efflux from the outer segment via a Na-Ca exchanger, suggesting a rise in internal free Ca in the light. We have now measured both Ca influx and efflux through the outer segment plasma membrane and find that, contrary to the calcium hypothesis, light seems to decrease rather than increase the free Ca concentration in the rod outer segment. This result implies that Ca does not mediate visual excitation but it probably has a role in light adaptation.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Retinopathy induced in mice by targeted disruption of the rhodopsin gene.

Retinitis pigmentosa (RP) represents the most common mendelian degenerative retinopathy of man, involving death of rod photoreceptors, cone cell degeneration, retinal vessel attenuation and pigmentary deposits. The patient experiences night blindness, usually followed by progressive loss of visual field. Genetic linkage between an autosomal dominant RP locus and rhodopsin, the photoreactive pigment of the rod cells, led to the identification of mutations within the rhodopsin gene in both dominant and recessive forms of RP. To better understand the functional and structural role of rhodopsin in the normal retina and in the pathogenesis of retinal disease, we generated mice carrying a targeted disruption of the rhodopsin gene. Rho-/- mice do not elaborate rod outer segments, losing their photoreceptors over 3 months. There is no rod ERG response in 8-week-old animals. Rho+/- animals retain the majority of their photoreceptors although the inner and outer segments of these cells display some structural disorganization, the outer segments becoming shorter in older mice. These animals should provide a useful genetic background on which to express other mutant opsin transgenes, as well as a model to assess the therapeutic potential of re-introducing functional rhodopsin genes into degenerating retinal tissues.

Age Factors↗
PubMed1983

Phospholipid domains in bovine retinal rod outer segment disk membranes.

Phospholipid behavior in bovine retinal rod outer segment disk membranes and in phosphatidylcholine membranes containing the photopigment rhodopsin is explored. 31P NMR spectra of these systems show two distinguishable resonances. One resembles closely the 31P NMR resonance normally obtained from phospholipid bilayers. The other resonance is much broader. Thus, there appear to be two phospholipid head-group domains in this retinal membrane. Each environment confers different properties on the head groups. Phosphatidylcholine membranes containing the disk photopigment also show two phospholipid domains. Therefore, the environment in the retinal rod outer segment disk membranes characterized by the broad resonance may arise from the influence of the integral membrane protein rhodopsin on the membrane phospholipid bilayer.

Animals↗
PubMed1980

Membrane assembly in retinal photoreceptors I. Freeze-fracture analysis of cytoplasmic vesicles in relationship to disc assembly.

To study precursor-product relationships between cytoplasmic membranes of the inner segment of photoreceptors and the continually renewed outer disc membrane, we have compared the density and size distribution of intramembrane particles (IMP) in various membrane compartments of freeze-fractured photoreceptor inner and outer segments. Both rod and cone outer segments of Xenopus laevis are characterized by a relatively uniform distribution of approximately 4,400-4,700 IMP/micron2 in P-face (PF) leaflets of disc membranes. A similar distribution of IMP is found in the outer segment plasma membrane, the ciliary plasma membrane, and in the plasma membrane of the inner segment in the immediate periciliary region. In each case the size distribution of IMP can be characterized as unimodal with a mean diameter of approximately 10 nm. PF leaflets of endoplasmic reticulum, Golgi complex, and vesicles near the cilium have IMP with a size distribution like that in the cilium and outer segment, but with an average density of approximately 2,000/micron2. In contrast, IMP are smaller in average size (approximately 7.5 nm) in PF leaflets of inner segment plasma membrane, exclusive of the periciliary rgion. The similarity of size distribution of IMP in inner segment cytoplasmic membranes and those within the plasmalemma of the cilium and outer segment suggest a precursor-product relationship between the two systems. The structure of the vesicle-rich periciliary region and the segregation of IMP with different size distributions in this region suggest that components destined for incorporation into the outer segment exist as preformed membrane packages (vesicles) which fuse with the inner segment plasma membrane in the periciliary region. Subsequently, membrane components may be transferred to forming discs of the outer segment via the ciliary plasma membrane.

Animals↗
PubMed1992

The influence of arrestin (48K protein) and rhodopsin kinase on visual transduction.

The shutoff of the phototransduction cascade in retinal rods requires the inactivation of light-activated rhodopsin. The underlying mechanisms were studied in functionally intact detached rod outer segments by testing the effect of either sangivamycin, an inhibitor of rhodopsin kinase, or phytic acid, an inhibitor of 48K protein binding to phosphorylated rhodopsin, on light responses recorded in whole-cell voltage clamp. The results suggest that isomerized rhodopsin is inactivated fully by multiple phosphorylation and that the binding of 48K protein accelerates recovery by quenching partially phosphorylated rhodopsin. Higher concentrations of sangivamycin cause changes in the light response that cannot be explained by selective inhibition of rhodopsin kinase and suggest that other protein kinases are needed for normal rod function.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

A method for the isolation of retinal pigment epithelial cells from adult rats.

PURPOSE: Isolating retinal pigment epithelium (RPE) cells from adult rat was the goal of this study. METHOD: A modification of the procedure of Mayerson et al was the method employed. After the cornea and lens were removed, the eyecup was treated with hyaluronidase plus collagenase. The neural retina was carefully removed with little RPE attached. The eyecup, free of retina, was treated with the same enzyme cocktail that loosens any attached rod outer segments as well as the attachment of the RPE to Bruch's membrane. RPE cells were isolated in calcium-free medium as sheets of cells. RESULTS: Light and electron microscopy revealed good cell morphology. Purity of the RPE preparation was established by microscopy, polyacrylamide gel electrophoresis, and lipid analysis. The viability of the isolated RPE was about 74% by trypan blue exclusion test. CONCLUSIONS: This technique enables RPE to be isolated from adult rat in quantities sufficient for biochemical analysis.

Animals↗
PubMed1989

Cholesterol heterogeneity in bovine rod outer segment disk membranes.

Rod outer segment disk membranes have been used to study visual transduction events. Numerous studies have also focused on protein-lipid interactions in these membranes. The possible heterogeneity of the disk membrane composition has not been addressed in such studies. Freeze fracture studies (Andrews, L. D., and Cohn, A. I. (1979) J. Cell Biol. 81, 215-220; Caldwell, R., and McLaughlin, B. (1985) J. Comp. Neurol. 236, 523-537) suggest a difference in cholesterol content between newly formed and old disks. This potential heterogeneity in disk membrane composition was investigated using digitonin. Osmotically intact bovine rod outer segment disk membranes prepared by Ficoll flotation were separated based on the cholesterol content of the disks. The addition of digitonin to disk membrane suspensions in a one-to-one molar ratio with respect to cholesterol produced an increase in the density of the membranes in proportion to the amount of cholesterol present. The digitonin-treated disks were separated into subpopulations using a sucrose density gradient. Disks were shown to vary in cholesterol to phospholipid ratio from 0.30 to 0.05. The ratio of phospholipid to protein remained constant in all disk subpopulations at approximately 65 phospholipids per protein. No significant change in the fatty acid composition of the disks was observed as a function of change in cholesterol content. This work demonstrates compositional heterogeneity in disk membranes which may ultimately affect function.

Animals↗
PubMed2000

Retinal degeneration in the nervous mutant mouse. III. Electrophysiological studies of the visual pathway.

The nervous (nr) mutation induces a progressive and severe degeneration of cerebellar Purkinje cells and retinal photoreceptors that is virtually complete within the first few months of life. Previous studies of the retina in nervous (nr/nr) mice have focused primarily on the structural abnormalities seen at the level of the photoreceptor cell bodies and outer segments. Here, we have carried out a series of functional studies of the visual pathway in nervous mice and have quantified the status of the inner retinal cell and plexiform layers. Affected animals were obtained by mating nr/+ heterozygotes and screening the offspring for the ataxia characteristic of nervous animals; phenotypically normal littermates (i.e. nr/+ or +/+) were used as controls. As described previously, there is a substantial loss of photoreceptors cells in the nervous retina and a marked shortening of the inner and outer segments. These changes are accompanied by a more modest decline in the thickness of the inner plexiform and inner nuclear layers. These anatomic abnormalities were accompanied by reproducible changes in visual function, as measured with the electroretinogram (ERG) and visual evoked potential (VEP). The dark-adapted ERGs of nervous and control mice had similar waveforms, although the nervous responses were substantially smaller in amplitude. The reductions in the amplitude of the ERG a-wave corresponded to the loss of photoreceptor cells and shortened outer segments seen histologically. Nevertheless, the kinetics of the leading edge of the a-wave did not differ between nervous and control mice, indicating that the rod outer segments of nervous mice continue to respond to light in a normal fashion. The amplitudes of cone ERGs were also reduced in nervous mice, although the extent of this reduction in any given animal was always less than that for rod-mediated ERG components. Overall, this result is consistent with cone involvement occurring only as a secondary effect of rod photoreceptor degeneration. The peak latencies of VEPs of nervous mice were slower than those of control littermates. These functional abnormalities correspond well to the structural changes induced by the nervous mutation, which does not appear to prevent visual signals from being transmitted centrally, beyond the limitations imposed by the degenerative process.

Animals↗
PubMed1986

A qualitative and quantitative analysis of the human fovea during development.

The anatomical development of the human fovea has been sampled from 22 weeks gestation to adulthood, using both qualitative and quantitative methods. The foveal depression continues to deepen after birth until 15 months, due to the migration of the cells of the inner retina toward the periphery. Before birth the rod-free zone or foveola is over 1000 microns in diameter, but it becomes progressively narrower after birth because of a centralward migration of cones. It reaches the adult diameter of 650-700 microns by 45 months of age. Postnatally, foveolar cone development is characterized by maturation, elongation, and an increase in packing density. Foveolar cone diameter changes markedly after birth, going from 7.5 microns at 5 days postnatal to 2 microns by 45 months. During this time the foveolar cone develops both its outer segment and basal axon process (fiber of Henle). This combination of elongation and centralward migration results in an increase of foveolar cone density from 18 cones/100 microns at 1 week postnatal to 42 cones/100 microns in the adult. Measures of foveola width and cone diameter reach the adult stage of development at 45 months of age, but the two important visual factors of outer segment length and cone packing density still are only half the adult values at 45 months of age.

Adult↗
PubMed1992

Ascorbate treatment prevents accumulation of phagosomes in RPE in light damage.

In dark-reared albino rats, exposure to 2 or 3 hr of intense light interrupted by 2 hr dark periods resulted in extensive degeneration of photoreceptor cells and degeneration of the retinal pigment epithelium (RPE). Ascorbate (ie, vitamin C) administration prior to light exposure protected photoreceptors and the RPE from light damage. In the present study, ascorbate-treated and untreated rats were exposed to various cycles of intermittent light. Immediately after this light exposure, phagosome frequency in the RPE was morphologically evaluated in comparable 50 microns sections. In untreated rats, exposure to 2 or 3 hr of intermittent light resulted in a five- to sixfold increase in phagosome density compared to unexposed controls. In contrast, no increase in phagosome density was observed in ascorbate-treated rats. In these animals, under all lighting regimens, phagosome levels remained essentially identical to those in rats not exposed to light. After a single nondamaging light exposure, phagosome density remained at the level of dark controls in ascorbate-treated and untreated rats. These results indicate that phagosome frequency may serve as an index for light damage and that the protective effect of ascorbate may be linked to its capacity to prevent rod outer segment shedding and phagocytosis under intense light conditions.

Animals↗
PubMed1992

Retinal pigment epithelial cells play a central role in the conservation of docosahexaenoic acid by photoreceptor cells after shedding and phagocytosis.

The involvement of retinal pigment epithelial (RPE) cells in the recycling of docosahexaenoic acid (DHA), from phagocytized disc membranes back to the retina, was studied in frogs subsequent to injection of [3H]DHA via the dorsal lymph sac. Rod outer segments (ROS) gradually accumulated [3H]DHA as a dense, heavily labeled region that arrived at the distal tips by 28 days post-injection. Autoradiographic analysis at the time of maximal shedding and phagocytosis (1-2 hr after the onset of light) showed diffusely (before 28 days) and heavily (after 28 days) labeled phagosomes in RPE cells. Biochemical analysis of the [3H]DHA-containing lipids of discs that contribute to the labeling of RPE cells after phagocytosis was also performed. Between 27 and 34 days, when 12% of retinal [3H]DHA-lipids present in disc membranes are phagocytized by RPE cells, total retinal labeling remained unchanged. Taken together, these data suggest that the [3H]DHA of the densely labeled region of the ROS was recycled back to the photoreceptor cells only after it had reached the RPE cells following 28 days post-injection. We conclude that, following daily phagocytosis of ROS tips, RPE cells play a central role in the conservation and redelivery of ROS-derived DHA back to photoreceptor cells through the interphotoreceptor matrix.

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↗
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↗
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↗