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PubMed2005

Nutritional manipulation of primate retinas. IV. Effects of n--3 fatty acids, lutein, and zeaxanthin on S-cones and rods in the foveal region.

Lutein and zeaxanthin are xanthophylls selectively accumulated by primate retinas that may protect the macula from age-related macular degeneration. In this project, we manipulated n-3 fatty acids, lutein and/or zeaxanthin levels in the diet and studied their possible outcome on S-cone and rod cell density in the foveal region. Rhesus monkeys (7-16 year, n=17) were fed from birth xanthophyll-free semipurified diets with either adequate or low n-3 fatty acids. Five monkeys were supplemented with lutein and six with zeaxanthin for 6-24 months, while six remained xanthophyll-free until sacrifice. Retinas were embedded in methacrylate and serial 2 microm sections were cut along the vertical meridian. Rod nuclei, and immuno-labelled outer segments of S-cones and rods, were reconstructed and counted in an 8 microm strip. The density profiles were compared with data from control monkeys (n=7) fed a standard laboratory diet. S-cone density profiles were symmetrical along the vertical meridian and the densities decreased rapidly with retinal eccentricity. Rod densities were higher in the superior region than the inferior region in most of the control and experimental animals. Unlike the significant effects observed for retinal pigment epithelial cells of these same monkeys (Leung, I.Y-F., Sandstrom, M.M., Zucker, C.L., Neuringer, M., Snodderly, D.M., 2004. Nutritional manipulation of primate retinas. II. Effects of age, n-3 fatty acids, lutein, and zeaxanthin on retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 45, 3244-3256), neither xanthophyll supplementation nor low dietary n-3 fatty acids produced consistent effects on S-cone or rod density profiles of the experimental animals. However, monkeys low in n-3 fatty acids had increased variability of S-cone density in the fovea and low density of foveal rod outer segments. The high variability suggests that the photoreceptors of some animals were resistant to the nutritional manipulations, while others may have been affected. Thus, the photoreceptors appear less sensitive than the retinal pigment epithelium to these nutritional manipulations. However, it is possible that more consistent effects would emerge at a later age or after exposure to stressors such as high light levels.

Animal Nutritional Physiological Phenomena↗
PubMed1994

The human photoreceptor membrane guanylyl cyclase, RetGC, is present in outer segments and is regulated by calcium and a soluble activator.

A human photoreceptor membrane guanylyl cyclase, RetGC, was recently cloned and expressed, but its localization and manner of regulation were not defined. We report here that RetGC is detected primarily in outer segments of human photoreceptor cells. Recombinant RetGC can be stimulated by a soluble retinal-specific factor. Ca2+ interferes with stimulation of RetGC by this factor with a cooperativity coefficient of 1.7 and EC50 near 200 nM. The Ca2+ sensitivities of recombinant RetGC and of guanylyl cyclase activity from rod outer segment membranes are very similar. Our findings indicate that RetGC is a photoreceptor-specific guanylyl cyclase which is stimulated by a retinal-specific activator and inhibited by physiologically relevant concentrations of free Ca2+. The Ca2+ sensitivity of RetGC may be responsible for some of the previously reported effects of Ca2+ on light adaptation and recovery of the dark state.

Amino Acid Sequence↗
PubMed1993

Fine structure of the retinal photoreceptors of the tiger salamander (Ambystoma tigrinum).

The retinal photoreceptors of the tiger salamander (Ambystoma tigrinum) have been studied by light and electron microscopy in both light- and dark-adaptation. Rods and cones are present in this duplex retina in a ratio of about 20:1. As in other urodele species these photoreceptors are very large cells. The rod outer segment is composed of bi-membranous discs of uniform diameter displaying several very deep incisures. The rod inner segment displays an ellipsoid of mitochondria and a myoid region which changes in diameter during the lighting cycle indicating that rods undergo photomechanical movements. Rod nuclei are located at all levels of the outer nuclear layer and rod spherules are large and display several invaginated and superficial synaptic sites. Cone photoreceptors while large cells are smaller than the rods. They show a smaller tapering outer segment, a large distal ellipsoid of mitochondria and a prominent paraboloid of glycogen but no oil droplet within the inner segment. Judging by the width of the myoid region which remains similar throughout the lighting cycle, cones in this species show no retino-motor responses. Cone nuclei are less electron dense than rods and are also located at all levels of the outer nuclear layer. The cone synaptic pedicle is larger than that of the rods and also shows several invaginated and superficial synaptic sites.

Adaptation, Ocular↗
PubMed1985

The effect of rhodopsin phosphorylation on the light-dependent activation of phosphodiesterase from bovine rod outer segments.

ATP quenches light-dependent phosphodiesterase (PDE) activation in rod outer segments presumably due to rhodopsin phosphorylation. Here we compared the efficiency of phosphorylated and non-phosphorylated rhodopsins as PDE activators in a reconstituted cell-free system. It is shown that the ability of phosphorylated membranes to activate this enzyme is suppressed compared with non-phosphorylated ones.

Animals↗
PubMed1982

Visual pigment and visual receptor cells in fetal and adult sheep.

The visual pigments, and the structure of the visual cells, were investigated by spectrophotometry and by light and electron microscopy in fetal and adult sheep. The rhodopsin system in adult sheep closely resembles that of cattle. The absorbance maxima of rhodopsin, lumirhodopsin, and metarhodopsin I are at 498, 490, and 480 nm, respectively. The estimated molar absorbance coefficient at the wavelength of maximum absorption of rhodopsin is 40,000M-1 . cm-1. Rhodopsin was detected from a fetal age of 85 days (term at 145 days). Partial bleaching of extracts from fetal eyes (95, 105, and 115 days) did not demonstrate cone pigments, although cones were present in fair numbers at a fetal age of about 105 days. The time course of rhodopsin formation between 85 and 140 days resembles a growth curve. The amount of rhodopsin shortly before birth (140 days) is about 0.6 times that in the adult. The number and dimensions of rod outer segments as well as packing of the discs were studied structurally and related to the rhodopsin content. A fairly good correlation was found at the earliest stages (95 and 105 days gestation age), when rhodopsin concentration was very low and rod outer segments were few and small, as well as at the latest stage (140 days). At 115 days the rhodopsin content observed by spectrophotometry was less than that indicated by the outer segment volume, probably mainly due to the outer segment discs and possibly to the rhodopsin molecules being less tightly packed than at 140 days.

Animals↗
PubMed1994

The photoreceptor G-protein transducin (Gt) is a substrate for ubiquitin-dependent proteolysis.

Bovine photoreceptor (rod outer segment) proteins were selectively degraded by an ATP- and ubiquitin-dependent mechanism in rabbit reticulocyte lysate and human retinal pigment epithelial cell supernatant. Proteolysis of 37, 40, 58, 68 and approximately 100 kDa proteins was accompanied by the formation of high mass (> 150 kDa) species (putative ubiquitin-protein conjugates. To identity degraded substrates, the photoreceptor GTP-binding protein, transducin (Gt alpha beta gamma), was purified by GTP elution. Degradation of transducin (> or = 50% loss of each subunit) by retinal pigment epithelial cell supernatant was ubiquitin-dependent (EC50 = 1.2 microM). Formation of high mass transducin species was coincident with degradation. These data identify a selective proteolytic mechanism that may regulate the photoreceptor GTP-binding protein.

Adenosine Triphosphate↗
PubMed1997

Functionally nonequivalent interactions of guanosine 5'-triphosphate, inosine 5'-triphosphate, and xanthosine 5'-triphosphate with the retinal G-protein, transducin, and with Gi-proteins in HL-60 leukemia cell membranes.

G-proteins mediate signal transfer from receptors to effector systems. In their guanosine 5'-triphosphate (GTP)-bound form, G-protein alpha-subunits activate effector systems. Termination of G-protein activation is achieved by the high-affinity GTPase [E.C. 3.6.1.-] of their alpha-subunits. Like GTP, inosine 5'-triphosphate (ITP) and xanthosine 5'-triphosphate (XTP) can support effector system activation. We studied the interactions of GTP, ITP, and XTP with the retinal G-protein, transducin (TD), and with G-proteins in HL-60 leukemia cell membranes. TD hydrolyzed nucleoside 5'-triphosphates (NTPs) in the order of efficacy GTP > ITP > XTP. NTPs eluted TD from rod outer segment disk membranes in the same order of efficacy. ITP and XTP competitively inhibited TD-catalyzed GTP hydrolysis. In HL-60 membranes, the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and leukotriene B4 (LTB4) effectively activated GTP and ITP hydrolysis by Gi-proteins. fMLP and LTB4 were at least 10-fold more potent activators of ITPase than of GTPase. Complement C5a effectively activated the GTPase of Gi-proteins but was only a weak stimulator of ITPase. The potency of C5a to activate GTP and ITP hydrolysis was similar. The fMLP-stimulated GTPase had a lower Km value than the fMLP-stimulated ITPase, whereas the opposite was true for the Vmax values. fMLP, C5a, and LTB4 did not stimulate XTP hydrolysis. Collectively, our data show that GTP, ITP, and XTP bind to G-proteins with different affinities, that G-proteins hydrolyze NTPs with different efficacies, and that chemoattractants stimulate GTP and ITP hydrolysis by Gi-proteins in a receptor-specific manner. On the basis of our results and the data in the literature, we put forward the hypothesis that GTP, ITP, and XTP act as differential signal amplifiers and signal sorters at the G-protein level.

Animals↗
PubMed1993

Modulation of the cGMP-gated channel of rod photoreceptor cells by calmodulin.

Photobleaching of rhodopsin in rod photoreceptors activates the visual cascade system leading to a decrease in cyclic GMP and the closure of cGMP-gated channels in the rod outer segment plasma membrane. Calcium plays an important role in the recovery of the rod outer segment to its dark state by regulating the resynthesis of cGMP by guanylate cyclase. Here we report that calmodulin, a Ca(2+)-binding protein present in the rod outer segment, increases the apparent Michaelis constant of the channel for cGMP. This results in a decrease in the rate of cation influx into the rod outer segment by two- to sixfold at low cGMP concentrations and has the effect of increasing the sensitivity of the channel to small changes in cGMP levels during phototransduction. Biochemical studies indicate that calcium-calmodulin binds to a protein of M(r) 240K which is tightly associated with the channel. On the basis of these studies, Ca2+ is suggested to play a central role in photorecovery and light adaptation, not only by regulating guanylate cyclase, possibly through recoverin, but also by modulating the cGMP-gated channel through calmodulin interaction with the 240K protein.

Animals↗
PubMed1993

Structural and biochemical identity of retinal rod outer segment membrane guanylate cyclase.

Recent molecular cloning reports show that there are at least three membrane guanylate cyclases in vertebrate retina: (1) atrial natriuretic factor receptor guanylate cyclase (ANF-RGC), (2) C-type natriuretic peptide receptor guanylate cyclase (CNP-RGC), and (3) "retinal guanylate cyclase" (RetGC). The specific cellular localization of the first two cyclases is unknown, but RetGC is apparently localized in photoreceptor cells, suggesting that it participates in visual transduction. With the overall objective of identifying the guanylate cyclase that is linked to phototransduction, we compared the structural and regulatory properties of the biochemically characterized 112 kDa bovine rod outer segment membrane guanylate cyclase (ROS-GC) with those of RetGC, ANF-RGC and CNP-RGC. The N-terminal and two internal peptide sequences of purified ROS-GC had about 90% similarity with the corresponding sequences of the RetGC; the sequence identity with natriuretic peptide receptor cyclases was about 30%. A 19 amino acid long sequence from a tryptic peptide of ROS-GC had no corresponding sequence in the other three cyclases. ROS-GC was inhibited by ATP but ANF-RGC and CNP-RGC were activated by ATP in the presence of the respective peptide hormones. These results suggest that ROS-GC represents a new subtype of the membrane guanylate cyclase family that is structurally and biochemically distinct from the other retinal cyclases.

Adenosine Triphosphate↗
PubMed1986

G-proteins of fat-cells. Role in hormonal regulation of intracellular inositol 1,4,5-trisphosphate.

Pertussis toxin abolishes hormonal inhibition of adenylate cyclase, hormonal stimulation of inositol 1,4,5-trisphosphate accumulation in rat fat-cells, and catalyses the ADP-ribosylation of two peptides, of Mr 39,000 and 41,000 [Malbon, Rapiejko & Mangano (1985) J. Biol. Chem. 260, 2558-2564]. The 41,000-Mr peptide is the alpha-subunit of the G-protein, referred to as Gi, that is believed to mediate inhibitory control of adenylate cyclase by hormones. The nature of the 39,000-Mr substrate for pertussis toxin was investigated. The fat-cell 39,000-Mr peptide was compared structurally and immunologically with the alpha-subunits of two other G-proteins, Gt isolated from the rod outer segments of bovine retina and Go isolated from bovine brain. After radiolabelling in the presence of pertussis toxin and [32P]NAD+, the electrophoretic mobilities of the fat-cell 39,000-Mr peptide and the alpha-subunits of Go and Gt were nearly identical. Partial proteolysis of these ADP-ribosylated proteins generates peptide patterns that suggest the existence of a high degree of homology between the fat-cell 39,000-Mr peptide and the alpha-subunit of Go. Antisera raised against purified G-proteins and their subunits were used to probe immunoblots of purified Gt, Gi, Go, and fat-cell membrane proteins. Although recognizing the 36,000-Mr beta-subunit band of Gt, Gi, Go and a 36,000-Mr fat-cell peptide, antisera raised against Gt failed to recognize either the 39,000- or the 41,000-Mr peptides of fat-cells or the alpha-subunits of Go and Gi. Antisera raised against the alpha-subunit of Go, in contrast, recognized the 39,000-Mr peptide of rat fat-cells, but not the alpha-subunit of either Gi or Gt. These data establish the identity of Go, in addition to Gi, in fat-cell membranes and suggest the possibility that either Go or Gi alone, or both, may mediate hormonal regulation of adenylate cyclase and phospholipase C.

Adenylate Cyclase Toxin↗
PubMed1989

Light exposure stimulates arachidonic acid metabolism in intact rat retina and isolated rod outer segments.

This paper presents evidence that short-term exposure to light increases synthesis of hydroxyeicosatetraenoic acid (HETE) and prostaglandin D2 (PGD2) and stimulates the uptake and metabolism of 20:4 in phospholipids and triacylglycerols in rat retina. There was a time-dependent increase in incorporation of 1-14C-20:4 into glycerolipids in both dark-adapted and light-exposed groups. Exposure to light for 15 or 30 min enhanced the acylation of 20:4 into phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and triacylglycerols. In the light-exposed groups there was a large increase in the conversion of 20:4 to leukotriene B4, two diHETEs, 5-HETE, 15-HETE, and PGD2. The stimulation of HETE synthesis by light could be due to early stages of light-induced lipid peroxidation in visual cells. To examine this, we studied peroxidation of 20:4 in isolated rod outer segments (ROS). There was more oxidation of 20:4 in light-exposed ROS, as compared to ROS incubated in the dark. Vitamin E and nordihydroguaiaretic acid inhibited the light-induced formation of some of these products. The data indicate that photo-oxidation of 20:4 in ROS is accompanied by enzymatic oxygenation that is stimulated by light. Increased production of HETEs an PGD2 may be a consequence of the light-induced stimulation of the metabolism of 20:4 in membrane phospholipids in the retina.

Animals↗
PubMed1992

Regulation of retinal cGMP cascade by phosducin in bovine rod photoreceptor cells. Interaction of phosducin and transducin.

Photoexcitation of retinal rod photoreceptor cells involves the activation of cGMP enzyme cascade in which sequential activation of rhodopsin, transducin, and the cGMP phosphodiesterase in the rod outer segment constitutes the signal amplification mechanism. Phosducin, a 33-kDa phosphoprotein, has been shown to form a tight complex with the T beta gamma subunit of transducin. In this study, we examined the interaction of phosducin-T beta gamma and the possible regulatory role of phosducin on the cGMP cascade. Addition of phosducin to photolyzed rod outer segment (ROS) membrane reduced the GTP hydrolysis activity of transducin as well as the subsequent activation of the cGMP phosphodiesterase. Phosducin also inhibited the pertussis toxin-catalyzed ADP-ribosylation of transducin, indicating that the interaction between the T alpha and T beta gamma subunits of transducin was interrupted upon binding of phosducin. The inhibitory effects of phosducin were reversed by the addition of exogenous T beta gamma. These results suggest that phosducin is capable of regulating the amount of T beta gamma available to interact with T alpha to form the active transducin complex and thereby functions as a negative regulator of the cGMP cascade. The phosducin-induced alteration of the subunit organization of transducin was examined by chemical cross-linking method using para-phenyl dimaleimide as cross-linker. It was found that the cross-linking among T alpha and T beta gamma was blocked in the presence of phosducin. This result implies that T beta gamma may undergo a conformational change upon phosducin binding which leads to the release of T alpha. Since phosducin is a soluble protein, the interaction with transducin only occurs when transducin is dissociated from ROS disc membrane. Indeed, phosducin failed to dissociate membrane-bound transducin and did not inhibit the initial cycle of transducin activation as measured by the presteady state GTP hydrolysis. However, phosducin interacts effectively with transducin released into solution after the initial activation and blocks the re-binding of T alpha. T beta gamma to ROS membrane by forming a tight complex with T beta gamma. This interaction may play an important role in regulating the turnover of the cGMP cascade in photoreceptor cells.

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