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PubMed1986

Phosphatidate phosphatase activity in isolated rod outer segment from bovine retina.

Phosphatidate phosphohydrolase (EC 3.1.3.4) was detected in isolated bovine rod outer segments and its properties investigated. The enzyme activity was assayed using aqueously dispersed 1,2-diacyl-sn-[2-3H]glycerol 3-phosphate as substrate. The phosphatidic acid concentration was optimal at 1 mM and the estimated Km value was 6.7 X 10(-4) M. The activity was linear for 60 min with a protein concentration of 0.3 mg. A clear pH optimum was observed at 7.5. When the enzyme activity was measured using a substrate phosphatidic acid containing 15% lyso compound, the production of diacylglycerols was inhibited by about 70-75% at all concentrations studied. In rod outer segment preparations containing 0.2 mM Mg2+, further additions of the ion (0.2-2.5 mM) only produced a slight inhibition of the activity at 2.5 mM. F- (50 mM), Ca2+ (1 mM) and EDTA (50 mM) inhibited the dephosphorylation of the substrate by 80, 10 and 70%, respectively. The aqueously dispersed phosphatidic acid-dependent activity present in rod outer segments was stimulated by Triton X-100 and taurocholate. Similar values were observed in the enzyme activity of entire rod outer segments and in that of disks obtained from them, showing the enzyme to be associated with disk membrane. The [3H]diacylglycerol production from [3H]phosphatidic acid was analyzed in synaptosomal-mitochondrial and microsomal fractions and in a crude pigment epithelium preparation. The degree of dephosphorylation of phosphatidic acid in these subcellular fractions was as follows: microsomes greater than synaptosomal-mitochondrial fraction greater than rod outer segment greater than crude pigment epithelium. The present report is the first evidence of phosphatidic acid phosphohydrolase activity associated with rod outer segment membranes.

Animals↗
PubMed1998

Phospholipase Cgamma1 in bovine rod outer segments: immunolocalization and light-dependent binding to membranes.

We have investigated the isozymes of a phosphoinositide-specific phospholipase C (PLC) in bovine retina using several monoclonal antisera to PLCbeta1, gamma1, and delta1. Immunoblot analysis showed that all three isozymes were present in the retina. Immunocytochemical localization in frozen bovine retina sections showed that PLCgamma1 was present in the photoreceptor cell layer, outer plexiform cell layer, inner plexiform cell layer, and ganglion cell layer. Immunoreaction within the photoreceptor cell layer was dependent on dark/light adaptation state of retinas. Immunoblot analysis of rod outer segments (ROS) with monoclonal or polyclonal antibodies to PLCgamma1 showed the presence of an immunoreactive band of 140 kDa. ROS prepared from retinas light-adapted in vitro had more PLCgamma1 on immunoblots than ROS from dark-adapted retinas. PLC enzyme activity in ROS from light-adapted retinas was 69 and 46% higher than ROS from dark-adapted retinas, when assayed in the presence and absence of ATP, respectively. This increase in enzyme activity was observed at [Ca2+]free between 0.32 and 100 microM. These results demonstrate the presence of PLCgamma1 in bovine ROS and show that ROS prepared from light-adapted retinas are enriched in this isozyme, suggesting that light may promote the binding of this isozyme to bleached ROS membranes.

Adaptation, Ocular↗
PubMed1986

Protein complement of rod outer segments of frog retina.

Rod outer segments (ROS) from frog retina have been purified by Percoll density gradient centrifugation, a procedure that preserves their form and intactness. One- and two-dimensional electrophoretic analysis reveals a smaller number of proteins than is observed in many cell organelles and permits quantitation of the 20 most abundant polypeptides. Rhodopsin accounts for 70% of the total protein (3 X 10(9) copies/outer segment), and approximately 70 other polypeptides are present at more than 6 X 10(4) copies/outer segment. Another 17% of the total protein is accounted for by the G-protein (3 X 10(8) copies/outer segment) that links rhodopsin bleaching and the activation of cyclic GMP phosphodiesterase (PDE). The phosphodiesterase accounts for 1.5% of the protein (1.5 X 10(7) copies/outer segment), and a 48,000-dalton component that binds to the membrane in the light accounts for a further 2.6%. The function of approximately 90% of the total protein in the outer segment is known, and two-thirds of the non-rhodopsin protein is accounted for by enzyme activities associated with cyclic GMP metabolism. The relative molar abundance of rhodopsin, G-protein, and PDE is 100:10:1. Apart from these major membrane-associated proteins, most of the other proteins are cytosolic. Thirteen other polypeptides are found at an abundance of one or more copies per 1000 rhodopsins, nine soluble and four membrane-bound, and their abundance relative to rhodopsin has been quantitated. ROS have been separated into subcellular fractions which resolve three classes of soluble, extrinsic membrane, and integral membrane proteins. A listing of the proteins that are phosphorylated and their subcellular localization is given. Approximately 25 phosphopeptides are detected, and most are in the soluble fraction. Fewer phosphorylated proteins are associated with the purified outer segments than with crude ROS. Distinct patterns of phosphorylation are associated with intact rods incubated with [32P]Pi and broken rods incubated with [gamma-32P]ATP.

Animals↗
PubMed1991

Acylation and glycosylation of rhodopsin in the rd mouse.

Retinas of 9-10-day-old rd and control mice were incubated for 2 hr with [14C]leucine along with either tritiated palmitic acid or galactose to investigate the acylation or glycosylation, respectively, of rhodopsin. Although other laboratories have reported that phosphorylation of rhodopsin is not detectable in rd retinas, the two post-translational modifications of rhodopsin investigated in the present work are detectable. The rod outer segments (ROS) were separated from the retinal debris containing the rough endoplasmic reticulum (RER) of photoreceptor cells by vortexing and then by linear sucrose gradients. The rhodopsin from the RER was purified by affinity chromatography and gel electrophoresis. In the acylation studies, the mean ratio of palmitate to leucine in the rd mouse was nearly twice that of controls (11.73 +/- 2.84 v. 6.81 +/- 1.04). Possible explanations for the disparity between the two groups could include: (1) a diminished internal pool size of the fatty acid; or (2) acylation of amino acids such as serine or threonine which normally are not acylated in rhodopsin. Treatment of purified rhodopsin with 1 M hydroxylamine released similar amounts of palmitate from the rd mice and controls. Hence, the higher ratio of palmitate to leucine in rd mice is apparently due to a diminished internal pool size. In the glycosylation studies, the ratio of galactose to leucine was very similar between rd mice and controls, 1.7 +/- 0.43 v. 2.47 +/- 0.74. Protein content and specific activity were determined for the crude ROS preparations and for the remaining retinal debris. Although the amount of ROS protein differed significantly between the two groups, the specific activities did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗
PubMed1994

Transforming growth factor-beta regulates human retinal pigment epithelial cell phagocytosis by influencing a protein kinase C-dependent pathway.

BACKGROUND: Transforming growth factor-beta (TGF-beta) plays an important role in the pathogenesis of many ocular diseases, including proliferative vitreoretinopathy. We examined the effect of TGF-beta on the phagocytosis of rod outer segments by retinal pigment epithelium (RPE), which is a major function of RPE, and investigated the dependence of this effect on the protein kinase C (PKC) pathway. METHODS: Phagocytotic uptake of fluoresceinated bovine rod outer segments was determined by flow cytometry. RPE cells were treated with TGF-beta 1 or TGF-beta 2 and their effects on phagocytosis were examined. The effects of various PKC inhibitors (calphostin C, staurosporine, and extended exposure to phorbol 12-myristate 13-acetate, PMA) and a stimulator (brief exposure to PMA) on RPE phagocytosis was evaluated. RESULTS: Both TGF-beta 1 and TGF-beta 2 up-regulated RPE phagocytosis and PMA abolished the up-regulating effect of TGF-beta. In contrast, PKC inhibition by staurosporine and calphostin C resulted in increased phagocytosis. A combination of TGF-beta and PKC inhibitor treatment did not produced any additive effect on phagocytosis. CONCLUSION: We concluded that TGF-beta up-regulates human RPE phagocytosis, but that this effect is counteracted by PKC activation. It is possible that this TGF-beta-induced effect is due, in part, to a negative modulation of the PKC-dependent pathway.

Alkaloids↗
PubMed1984

Retinitis pigmentosa: a quantitative study of the apical membrane of normal and dystrophic human retinal pigment epithelial cells in tissue culture in relation to phagocytosis.

The phagocytic capabilities of both normal and dystrophic human retinal pigment epithelial cells were assessed by challenging cultures with both non-biological and biological particles. Cells from either source ingested both carbon particles and latex microspheres. Quantification of the phagocytic process in relation to microspheres showed that uptake was linear over an 8 h period and the rate was dose-dependent. All cultures phagocytosed rod outer segments from a variety of species, but excluded bacteria. By using bovine rod outer segments labelled with [H3] choline both normal and dystrophic cells were still accumulating label 24 h subsequent to challenge; however, this technique had significant problems. The introduction of any particulate matter into the culture medium resulted in changes in the apical membrane of the recipient cells. Such responses were quantified in terms of apical projections per unit area. Counts were always greater in cells challenged with biological material even if the material was not engulfed.

Aged↗
PubMed1989

Evidence that microtubules do not mediate opsin vesicle transport in photoreceptors.

The organization of the rod photoreceptor cytoskeleton suggests that microtubules (MTs) and F actin are important in outer segment (OS) membrane renewal. We studied the role of the cytoskeleton in this process by first quantifying OS membrane assembly in rods from explanted Xenopus eyecups with a video assay for disc morphogenesis and then determining if the rate of assembly was reduced after drug disassembly of either MTs or F actin. Membrane assembly was quantified by continuously labeling newly forming rod OS membranes with Lucifer Yellow VS (LY) and following the tagged membranes' distal displacement along the OS. LY band displacement displayed a linear increase over 16 h in culture. These cells possessed a longitudinally oriented network of ellipsoid MTs between the sites of OS protein synthesis and OS membrane assembly. Incubation of eyecups in nocodazole, colchicine, vinblastine, or podophyllotoxin disassembled the ellipsoid MTs. Despite their absence, photoreceptors maintained a normal rate of OS assembly. In contrast, photoreceptors displayed a reduced distal displacement of LY-labeled membranes in eyecups treated with cytochalasin D, showing that our technique can detect drug-induced changes in basal rod outer segment assembly. The reduction noted in the cytochalasin-treated cells was due to the abnormal lateral displacement of newly added OS disc membranes that occurs with this drug (Williams, D. S., K. A. Linberg, D. K. Vaughan, R. N. Fariss, and S. K. Fisher. 1988. J. Comp. Neurol. 272:161-176). Together, our results indicate that the vectorial transport of OS membrane constituents through the ellipsoid and their assembly into OS disc membranes are not dependent on elliposid MT integrity.

Animals↗
PubMed1991

The NH2-terminal alpha subunit attenuator domain confers regulation of G protein activation by beta gamma complexes.

Gs and Gi, respectively, activate and inhibit the enzyme adenylyl cyclase. Regulation of adenylyl cyclase by the heterotrimeric Gs and Gi proteins requires the dissociation of GDP and binding of GTP to the alpha s or alpha i subunit. The beta gamma subunit complex of Gs and Gi functions, in part, to inhibit GDP dissociation and alpha subunit activation by GTP. Multiple beta and gamma polypeptides are expressed in different cell types, but the functional significance for this heterogeneity is unclear. The beta gamma complex from retinal rod outer segments (beta gamma t) has been shown to discriminate between alpha i and alpha s subunits (Helman et al: Eur J Biochem 169:431-439, 1987). beta gamma t efficiently interacts with alpha i-like G protein subunits, but poorly recognizes the alpha s subunit. beta gamma t was, therefore, used to define regions of the alpha i subunit polypeptide that conferred selective regulation compared to the alpha s polypeptide. A series of alpha subunit chimeras having NH2-terminal alpha i and COOH-terminal alpha s sequences were characterized for their regulation by beta gamma t, measured by the kinetics of GTP gamma S activation of adenylyl cyclase. A 122 amino acid NH2-terminal region of the alpha i polypeptide encoded within an alpha i/alpha s chimera was sufficient for beta gamma t to discriminate the chimera from alpha s. A shorter 54 amino acid alpha i sequence substituted for the corresponding NH2-terminal region of alpha s was insufficient to support the alpha i-like interaction with beta gamma t. The findings are consistent with our previous observation (Osawa et al: Cell 63:697-706, 1990) that a region in the NH2-terminal moiety functions as an attenuator domain controlling GDP dissociation and GTP activation of the alpha subunit polypeptide and that the attenuator domain is involved in functional recognition and regulation by beta gamma complexes.

Adenylyl Cyclases↗
PubMed1990

Retinal detachment prevents normal assembly of disk membranes in vitro.

The effects of retinal detachment upon disk membrane assembly in rod outer segments were assessed in Xenopus laevis retinas that had been maintained in eyecup cultures for up to 4 days. In these cultures, assembly of disk membranes occurred at a normal rate in regions of the retina that remained attached to the retinal pigment epithelium. In regions of the retina that were detached from the pigment epithelium, the assembly of new disk membranes either was abnormal or was inhibited. This result cannot be attributed to reduced access of cells in the detached retina to oxygen and metabolites. The experiments described here suggest that the apposition of the retina with the pigment epithelium is a necessary condition for normal disk membrane assembly in Xenopus retinas. This effect may be mediated by contact between the rod outer segments and the pigment epithelium, or by trophic factors in the subretinal space.

Animals↗
PubMed2002

Tamoxifen induces changes in the lipid composition of the retinal pigment epithelium cell line D407.

Tamoxifen, the antioestrogenic drug prescribed for long-term, low-dose therapy of breast cancer, induces retinopathy. This study evaluates the effects of tamoxifen on the human retinal pigment epithelial cell line D407, attempting to identify the underlying mechanisms on tamoxifen-induced retinopathy and the involvement of cellular membranes in the cytotoxic action mechanism. We demonstrate that the tamoxifen-induced decrease in the cell growth of the D407 cell line results from pyknosis and cell cycle arrest rather than from necrosis. Furthermore, D407 cells influence the lipid composition of both plasma membrane and intracellular membranes in response to tamoxifen. Tamoxifen increases the physical order of the lipid bilayer. We observed a compensatory decrease in the cholesterol content of the plasma membrane which results in an increase of the plasma membrane fluidity. In intracellular membranes the phosphatidylcholine content is reduced to 50% of the controls. This reduction may be related to the formation of a second messenger via phospholipase pathway and sustained activation of protein kinase C. Since increased plasma membrane fluidity as well as sustained activation of protein kinase C influence the rod outer segments binding and/or ingestion by retinal pigment epithelial cells, our results suggest that membrane-mediated pathways contribute to the tamoxifen-induced retinopathy.

Antineoplastic Agents, Hormonal↗
PubMed2000

Retinal vascular changes induced by the oxidative stress of alpha-tocopherol deficiency contrasted with diabetic microangiopathy.

It has been proposed that oxidative tissue damage is involved in the development of diabetic angiopathies. To evaluate this hypothesis, experiments were conducted to identify the retinal vessel changes induced by the oxidative stress related to alpha-tocopherol deficiency and examine possible similarities with the lesions characteristic of diabetic retinopathy. Twenty-one-day-old male Fisher 344 albino rats were divided randomly to receive a basal, chemically defined diet either with (adequate group) or without (deficient group) alpha-tocopherol. After 6 and 8 months, some rats (n = 3 per group) were killed and the eyes removed. In order to evaluate cell integrity and localization of lipofuscin-specific autofluorescence by light and fluorescence microscopy, some of the retinas were prepared for cryostat-sections while others were digested by elastase to isolate intact retinal vasculatures. After 8 and 14 months, the central retina of one eye per rat (n = 6 to 8 per group) was examined by electron microscopy for retinal capillary basement membrane (RCBM) thickening and other ultrastructural changes. At 6 and 8 months, the deficient rats exhibited extensive shortening and disarray of rod outer segments (ROS), marked loss of photoreceptor cells, and pronounced increases in the numbers of granules with lipofuscin-specific autofluorescence in the retinal pigment epithelium (RPE) and retinal vessels. At 14 months, the ultrastructure revealed that the damage to ROS involved disruption of membranes and that the capillary lipofuscin was contained mainly within the endothelial cells. Membrane remnants were found in the lipofuscin granules of both the RPE and retinal vessels. In addition, there was an increase in RCBM thickness (98.7 +/- 2.6 nm vs. 86.9 +/- 2.9 nm). RCBM thickening was the only finding common with diabetic retinopathy, and the thickening was 13.6%, significantly less than that reported in diabetic rat models with 8 and 14 months durations (34% and 53.1%, respectively). Capillary lipofuscin accumulation, which was prominent in the deficient rats, is not notable in diabetes. Both the moderate RCBM thickening and marked lipofuscin accumulations seen in alpha-tocopherol-deficient rats were similar to changes occurring in the aging process, though more pronounced. The spectrum of microangiopathies characteristic of diabetic retinopathy did not develop in alpha-tocopherol-deficient rats. These findings suggest that oxidative damage, though probably involved, is unlikely to play a predominant role in the development of diabetic retinal microangiopathies.

Animals↗
PubMed1994

Superoxide inhibits proliferation and phagocytic internalization of photoreceptor outer segments by bovine retinal pigment epithelium in vitro.

Experiments were performed to investigate the effect of free radical damage on two aspects of retinal pigmented epithelium (RPE) metabolism, namely, proliferation and phagocytosis. Bovine RPE cells were maintained in monolayer cultures, either as passaged (for proliferation and lysosomal activity assays) or primary cultures (for phagocytosis measurements). Free radicals (superoxide anions) were generated by a xanthine oxidase (XO)-hypoxanthine (HX) reaction. Total phagocytosis (binding plus ingestion of rod outer segments (ROS)) was quantitated by radioimmunoassay using a specific anti-opsin antibody and iodinated secondary antibody. In some cases, agents with known or possible protective influences against oxidative damage, i.e., superoxide dismutase (SOD), vitamin E, and basic fibroblast growth factor (bFGF), were tested for their activity in this model system. RPE cell proliferation was inhibited in a HX-XO dose-dependent manner, in the absence of cell toxicity. Modifications of cell morphology were also noticed. Either simultaneous exposure of RPE cells to ROS membranes and HX-XO or pretreatment of ROS membranes with HX-XO prior to their addition to RPE monolayers led to a statistically significant 20-30% decrease in phagocytosis relative to control values. This decrease was essentially observed in the binding phase of phagocytosis, indicating damage to ROS surface molecules as the primary event. Addition of SOD or vitamin E prevented this loss of phagocytic activity, whereas bFGF had no effect. Superoxide radicals did not, however, affect phagocytosis when RPE cells were exposed to them alone, prior to incubation with ROS; nor did they alter a later stage in the phagocytic process, acid phosphatase activity. This tissue culture model represents a convenient system for analyzing free radical damage in different aspects of RPE-photoreceptor behavior and may be useful in studying this phenomenon in several retinal disorders.

Acid Phosphatase↗
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

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↗
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↗