A high content, small molecule screen identifies candidate molecular pathways that regulate rod photoreceptor outer segment renewal.

The outer segment of the vertebrate rod photoreceptor is a highly modified cilium composed of many discrete membranous discs that are filled with the protein machinery necessary for phototransduction. The unique outer segment structure is renewed daily with growth at the base of the outer segment where new discs are formed and shedding at the distal end where old discs are phagocytized by the retinal pigment epithelium. In order to understand how outer segment renewal is regulated to maintain outer segment length and function, we used a small molecule screening approach with the transgenic (hsp70:HA-mCherryTM) zebrafish, which expresses a genetically-encoded marker of outer segment renewal. We identified compounds with known bioactivity that affect five content areas: outer segment growth, outer segment shedding, clearance of shed outer segment tips, Rhodopsin mislocalization, and differentiation at the ciliary marginal zone. Signaling pathways that are targeted by the identified compounds include cyclooxygenase in outer segment growth, γ-Secretase in outer segment shedding, and mTor in RPE phagocytosis. The data generated by this screen provides a foundation for further investigation of the signaling pathways that regulate photoreceptor outer segment renewal.

New findings in ATP supply in rod outer segments: insights for retinopathies.

BACKGROUND INFORMATION: The rod outer segment (OS) is the specialised organelle where phototransduction takes place. Our previous proteomic and biochemical analyses on purified rod disks showed the functional expression of the respiratory chain complexes I-IV and F1 Fo -ATP synthase in OS disks, as well as active soluble tricarboxylic acid cycle enzymes. Here, we focussed our study on the whole OS that contains the cytosol and plasma membrane and disks as native flattened saccules, unlike spherical osmotically intact disks. RESULTS: OS were purified from bovine retinas and characterised for purity. Oximetry, ATP synthesis and cytochrome c oxidase (COX) assays were performed. The presence of COX and F1F0-ATP synthase (ATP synthase) was assessed by semi-quantitative Western blotting, immunofluorescence or confocal laser scanning microscopy on whole bovine retinas and bovine retinal sections and by immunogold transmission electron microscopy (TEM) of purified OS or bovine retinal sections. Both ATP synthase and COX are catalytically active in OS. These are able to consume oxygen (O2) in the presence of pyruvate and malate. CLSM analyses showed that rhodopsin autofluorescence and MitoTracker Deep Red 633 fluorescence co-localise on rod OS. Data are confirmed by co-localisation studies of ATP synthase with Rh in rod OS by immunofluorescence and TEM in bovine retinal sections. CONCLUSIONS: Our data confirm the expression and activity of COX and ATP synthase in OS, suggestive of the presence of an extra-mitochondrial oxidative phosphorylation in rod OS, meant to supply ATP for the visual transduction. In this respect, the membrane rich OS environment would be meant to absorb both light and O2. The ability of OS to manipulate O2 may shed light on the pathogenesis of many retinal degenerative diseases ascribed to oxidative stress, as well as on the efficacy of the treatment with dietary supplements, presently utilised as supporting therapies.

Formation of lipofuscin-like material in the RPE Cell by different components of rod outer segments.

The mechanisms that control the natural rate of lipofuscin accumulation in the retinal pigment epithelial (RPE) cell and its stability over time are not well understood. Similarly, the contributions of retinoids, phospholipids and oxidation to the rate of accumulation of lipofuscin are uncertain. The experiments in this study were conducted to explore the individual contribution of rod outer segments (ROS) components to lipofuscin formation and its accumulation and stability over time. During the period of 14 days incubation of ROS, lipofuscin-like autofluorescence (LLAF) determined at two wavelengths (530 and 585 nm) by fluorescence-activated cell sorting (FACS) was measured from RPE cells. The autofluorescence increased in an exponential manner with a strong linear component between days 1 and 7. The magnitude of the increase was larger in cells incubated with 4-hydroxynonenal (HNE-ROS) compared with cells incubated with either bleached or unbleached ROS, but with a different spectral profile. A small (10-15%) decrease in LLAF was observed after stopping the ROS feeding for 14 days. The phagocytosis rate of HNE-ROS was higher than that of either bleached or unbleached ROS during the first 24 h of supplementation. Among the different ROS components, the increase of LLAF was highest in cells incubated with all-trans-retinal. Surprisingly, incubation with 11-cis-retinal and 9-cis-retinal also resulted in strong LLAF increase, comparable to the increase induced by all-trans-retinal. Supplementation with liposomes containing phosphatidylethanolamine (22: 6-PE) and phosphatidylcholine (18:1-PC) also increased LLAF, while incubation with opsin had little effect. Cells incubated with retinoids demonstrated strong dose-dependence in LLAF increase, and the magnitude of the increase was 2-3 times higher at 585 nm compared to 530 nm, while cells incubated with liposomes showed little dose-dependence and similar increase at both wavelengths. Very little difference in LLAF was noted between cells incubated with either unbleached or bleached ROS under any conditions. In summary, results from this study suggest that supplementation with various ROS components can lead to an increase in LLAF, although the autofluorescence generated by the different classes of components has distinct spectral profiles, where the autofluorescence induced by retinoids results in a spectral profile closest to the one observed from human lipofuscin. Future fluorescence characterization of LLAF in vitro would benefit from an analysis of multiple wavelengths to better match the spectral characteristics of lipofuscin in vivo.

Lipid differences in rod outer segment membranes of rats with P23H and S334ter opsin mutations.

PURPOSE: Retinal degenerations and diets low in n-3 fatty acids are associated with decreased docosahexaenoic acid (22:6n-3) in retina and plasma and with sterol abnormalities in retina and sperm. Using wild type (WT) and transgenic rats with P23H and S334ter opsin mutations, we evaluated retinal cholesterol levels, cholesterol synthesis, and fatty acid compositions of phospholipid classes in animals fed diets enriched in n-3 or n-6 polyunsaturated fatty acids. METHODS: Pregnant WT and heterozygous P23H and S334ter transgenic (TG) rats were fed safflower (safflower oil [SO], high n-6, trace n-3 fatty acids) or flaxseed oil (flaxseed oil [FO], high n-3, moderate n-6 fatty acids) diets beginning at E15, and pups were continued on the diets after weaning. Rod outer segment (ROS) membranes were prepared from 55-day-old rats, and the ratios of total fatty acid to cholesterol and the fatty acid compositions of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) in ROS were determined. Intravitreal injections of [3H]acetate were given to 35-day-old WT and TG rats fed standard chow-diets. Endogenous cholesterol mass and de novo [3H]cholesterol synthesis were measured and normalized to total ROS fatty acid content. Multivariate analysis of variance (ANOVA) with post hoc Newman-Keuls tests were used to determine statistical differences. RESULTS: The relative levels of PC, PE, and PS were similar in all three rat strains independent of diet. Total lipids, PC, PS, and PE of ROS FO fed rats had higher levels of 22:6n-3 and lower levels of 22:5n-6 than those fed SO. Rats fed SO had higher levels of 22:5n-6 than those fed FO. Significant increases in 18:1n-9 were seen in PC and PS of P23H and S334ter rats; arachidonate (20:4n-6) increased only in PE. These changes were independent of diet. ROS membranes of transgenic rats were cholesterol enriched, relative to WT ROS, yet retinal cholesterol synthesis was not altered. Plasma cholesterol levels of transgenic rats were not different from those of WT rats. CONCLUSIONS: Endogenous levels of cholesterol, 18:1n-9, 20:4n-6, 22:5n-6, and 22:6n-3 were altered in ROS membranes of P23H and S334ter compared to WT rats. There appear to be two pools of 22:6n-3 in rat ROS, one that is sensitive to retinal degenerations and one that is not. The stress induced reduction in 22:6n-3 was not specific to any phospholipid class and was not caused by alteration of relative amounts of PC, PS, or PE in the membrane. Elevated retinal cholesterol may be a result of either an increased half life or an increased uptake of cholesterol from the blood.

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.

P23H and S334ter opsin mutations: Increasing photoreceptor outer segment n-3 fatty acid content does not affect the course of retinal degeneration.

PURPOSE: The n-3 polyunsaturated fatty acids (PUFA) facilitate retinal development and function. Rats carrying transgenes with P23H and S334ter rhodopsin mutations lose their photoreceptors and have lower levels of 22:6n-3 in rod photoreceptor outer segments (ROS) than wild type (WT) animals. We tested the hypothesis that the rate of retinal degeneration in these mutant animals could be sensitive to the n-3 fatty acid content of retina. METHODS: Beginning embryonic day 15, WT and heterozygous transgenic rats with P23H and S344ter rhodopsin mutations were fed semi-synthetic diets enriched in n-6 (safflower oil, SO) or n-3 (flaxseed oil, FO) PUFA. At 35 and 55 days of age, electroretinographic (ERG) response, outer nuclear layer (ONL) thickness, and fatty acid composition of plasma and ROS were determined. Student's t-tests and multivariate analysis of variance with post hoc tests determined statistical differences. RESULTS: Rats fed FO or SO diets had different n-6/n-3 PUFA ratios in plasma (1.3 and 62) and ROS (0.2 and 1.1, respectively). Although there were profound effects of the diets on the plasma fatty acid composition, there were only minor differences between WT and transgenic animals within each dietary regime. The ROS of FO fed rats had 70% more 22:6n-3 than those fed SO, and the WT had higher concentrations of 22:6n-3 than the transgenic animals (WT>P23H>S334ter). In contrast, there was no difference in 22:6n-3 levels in ROS of WT and transgenic rats fed the SO diet. At P55, both transgenic lines had diminished ERGs and ONL thickness relative to the WT. There was no detectable effect of ROS fatty acid enrichment on the rate of retinal degeneration in the transgenic animals. However, the FO-diet provided a modest protection of function (b-wave) in S334ter animals. CONCLUSIONS: Feeding n-3 fatty acids to rats with mutant rhodopsin transgenes significantly increased the levels of 22:6n-3 in ROS membranes, but had no effect on the rate of retinal degeneration. Therefore, the degeneration is not the result of low (or high) 22:6n-3 in ROS and supplementation with 18:3n-3 will not rescue dying photoreceptor cells in these animal models of inherited retinal degenerations.

Structure and Ca2+ regulation of frog photoreceptor guanylate cyclase, ROS-GC1.

Rod outer segment membrane guanylate cyclase (ROS-GC) is a critical component of the vertebrate phototransduction machinery. In response to photoillumination, it senses a decline in free Ca(2+) levels from 500 to below 100 nM, becomes activated, and replenishes the depleted cyclic GMP pool to restore the dark state of the photoreceptor cell. It exists in two forms, ROS-GC1 and ROS-GC2. In outer segments, ROS-GCs sense fluctuations in Ca(2+) via two Ca(2+)-binding proteins, which have been termed GCAP1 and GCAP2. In the present study we report on the cloning of two ROS-GCs from the frog retinal cDNA library. These cyclases are the structural and functional counterparts of the mammalian ROS-GC1 and ROS-GC2. There is, however, an important difference between the regulation of mammalian and frog ROS-GC1: In contrast to the mammalian, the frog form does not require the myristoylated form of GCAP1 for its Ca(2+)-dependent modulation. This feature is not dependent upon the ability of frog GCAP1 to bind Ca(2+) because unmyristoylated GCAP1 mutants which do not bind Ca(2+), activate frog ROS-GC1. The findings establish frog as a suitable phototransduction model and show a facet of frog ROS-GC signaling, which is not shared by the mammalian form.

Identification of novel genes and altered signaling pathways in the retinal pigment epithelium during the Royal College of Surgeons rat retinal degeneration.

Shed photoreceptor outer segments (POS) are phagocytosed by RPE cells in a circadian manner. The homozygous deletion of the c-mer gene abolishes the ingestion phase of this phagocytosis in the Royal College of Surgeons (RCS) rat strain, which in turn leads to the death of photoreceptor cells. We identified RPE transcripts for which the expression is modulated by the abrogation of POS phagocytosis. A microarray approach and the differential display (DDRT-PCR) technique revealed 116 modulated known genes, 4 modulated unknown genes, and 15 expressed sequenced tags (ESTs) corresponding to unknown genes. The microarray and DDRT-PCR analyses detected alterations in signaling pathways such as the phosphatidylinositol 3-kinase-Akt-mTOR pathway and the DLK/JNK/SAPK pathway. The abrogation of POS phagocytosis caused a decrease in endomembrane biogenesis and altered endocytosis, exocytosis, transcytosis, and several metabolic and signaling pathways in RCS RPE cells. We also found differential levels of transcripts encoding proteins involved in phagocytosis, vesicle trafficking, the cytoskeleton, retinoic acid, and general metabolism.

Effect of dietary n-3 fatty acids on the composition of long- and very-long-chain polyenoic fatty acid in rat retina.

The effect of dietary supplementation with n-3 fatty acids, primarily docosahexaenoic acid (DHA) with high purity, on the fatty acid composition, especially very-long-chain fatty acids (VLCFA) longer than DHA, with four or six double bonds, in the rod outer segment (ROS) membranes of young Sprague-Dawley rats was investigated. After several weeks of feeding, diets high in n-3 fatty acids increased the DHA level significantly, while there were decreased levels of most n-6 fatty acids, such as arachidonic acid and 22:5n-6. Six kinds of VLCFA were detected by gas chromatography-mass spectrometry (GC-MS). Feeding a high n-3 fatty acid diet significantly increased the content of some n-3 VLCFAs such as 26:4n-3 and 30:4n-3 in ROS membranes, but not all detected n-3 VLCFAs. This study demonstrates that the dietary level of n-3 fatty acids not only affects the level of DHA, but also the levels of VLCFA in ROS membranes.

Effect of dietary DHA on DHA levels in retinal rod outer segments in young versus mature rats.

We compared the effect of direct supplementation with docosahexaenoic acid (DHA) on the fatty acid composition of the liver and the rod outer segment (ROS) membranes of the retina in young (five-week-old) and mature (one-year-old) rats. In young rats, a high content of DHA in the diet (9.7% of total energy) effectively increased the proportion of DHA in ROS membranes (41.8%), compared with the proportion observed in a linoleic acid (LA) diet group (control, 31.6%). The proportion of DHA was also significantly higher in the livers of young DHA-fed rats. These results show that direct supplementation with DHA is very effective in increasing DHA levels in the ROS membranes and livers of developing animals. In contrast, in mature rats there was no significant increase in the proportion of DHA in the ROS membranes, even after the highest dose (8.4% of total energy) of DHA, although the proportion of DHA was significantly higher in the livers of DHA-fed rats. The changes in fatty acid composition in the ROS membranes were different in young and mature rats fed high-DHA diets. Our findings indicate that mature rats maintain a constant level of DHA in the ROS membranes even after being directly supplemented with high doses of DHA.

Functional interaction between the two halves of the photoreceptor-specific ATP binding cassette protein ABCR (ABCA4). Evidence for a non-exchangeable ADP in the first nucleotide binding domain.

ABCR, also known as ABCA4, is a member of the superfamily of ATP binding cassette transporters that is believed to transport retinal or retinylidene-phosphatidylethanolamine across photoreceptor disk membranes. Mutations in the ABCR gene are responsible for Stargardt macular dystrophy and related retinal dystrophies that cause severe loss in vision. ABCR consists of two tandemly arranged halves each containing a membrane spanning segment followed by a large extracellular/lumen domain, a multi-spanning membrane domain, and a nucleotide binding domain (NBD). To define the role of each NBD, we examined the nucleotide binding and ATPase activities of the N and C halves of ABCR individually and co-expressed in COS-1 cells and derived from trypsin-cleaved ABCR in disk membranes. When disk membranes or membranes from co-transfected cells were photoaffinity labeled with 8-azido-ATP and 8-azido-ADP, only the NBD2 in the C-half bound and trapped the nucleotide. Co-expressed half-molecules displayed basal and retinal-stimulated ATPase activity similar to full-length ABCR. The individually expressed N-half displayed weak 8-azido-ATP labeling and low basal ATPase activity that was not stimulated by retinal, whereas the C-half did not bind ATP and exhibited little if any ATPase activity. Purified ABCR contained one tightly bound ADP, presumably in NBD1. Our results indicate that only NBD2 of ABCR binds and hydrolyzes ATP in the presence or absence of retinal. NBD1, containing a bound ADP, associates with NBD2 to play a crucial, non-catalytic role in ABCR function.

Alterations in retinal rod outer segment fatty acids and light-damage susceptibility in P23H rats.

PURPOSE: To determine whether dietary-induced alterations in the long-chain polyunsaturated fatty acid content of retinal rod outer segments (ROS) of P23H rats, a transgenic model of retinitis pigmentosa (RP), prolongs photoreceptor cell life. METHODS: Heterozygous P23H and normal Sprague-Dawley rats were fed a standard house diet or a diet deficient in 18:3n-3. Diet-deficient rats were given supplements of either linseed oil (high in 18:3n-3) or fish oil (high in 20:5n-3). ROS fatty acid profiles and serum fatty acids were determined by gas chromatography. Serum cholesterol was evaluated by HPLC. Retinal damage was assessed by measuring whole-retina rhodopsin and DNA content before and after exposure to high-intensity light. RESULTS: The retinas of 60 day old, cyclic-light-reared, P23H transgenic rats contained 50% of the rhodopsin and 75% of the DNA content found in control Sprague-Dawley rats. Eight hours of intense light had little effect on the rhodopsin or DNA content in the Sprague-Dawley rats, but resulted in rhodopsin and DNA losses of nearly 70%, compared to controls, in P23H animals fed either a standard or an 18:3n-3-deficient diet. Supplementation with linseed oil resulted in small, statistically insignificant, increases in the rhodopsin and DNA losses, which occurred after exposure to intense light, in P23H transgenics. In unexposed animals, supplementation with linseed oil or fish oil had no effect on either rhodopsin or DNA levels in P23H rats or in Sprague-Dawley controls. On standard diet, the ROS 22:6n-3 (DHA) content in P23H rats was lower than that of control animals. DHA decreased in both groups when an 18:3-deficient diet was fed. The reduction was greater in controls than in P23H transgenics, but a concomitant increase in 22:5n-6 was nearly the same in both groups. Supplementation of the 18:3-deficient diet with linseed oil or fish oil in P23H rats resulted in a ROS fatty acid profile comparable to that of Sprague-Dawley rats raised on a standard diet. Serum DHA and 22:5n-6 levels were low in both groups. No significant differences in serum cholesterol were observed as a function of genotype or diet. CONCLUSIONS: Heterozygous P23H rats are capable of forming ROS DHA from dietary fatty acid precursors found in linseed oil (18:3n-3) or fish oil (20:5n-3). Under all dietary conditions, P23H transgenics are highly susceptible to retinal damage from exposure to intense light. Although levels of DHA in the ROS of P23H rats could be altered by dietary manipulation, only small changes in photoreceptor cell survival, as measured by whole-retina rhodopsin and DNA content, were observed. The lower-than-normal levels of ROS DHA may reflect an adaptive, possibly protective, mechanism in the P23H transgenic rat model of RP.

Transport to the photoreceptor outer segment by myosin VIIa and kinesin II.

This paper reviews our studies in which we have used mutant mice to investigate the roles of myosin VIIa and kinesin II in the transport of proteins to the photoreceptor outer segment. These studies suggest that both motors participate in moving opsin along the connecting cilium. Given the velocities measured for these motors in vitro, it is predicted that the resulting concentration of opsin in the plasma membrane of the connecting cilium is surprisingly low.

Retinal pigment epithelial acid lipase activity and lipoprotein receptors: effects of dietary omega-3 fatty acids.

PURPOSE: To show that fish oil-derived omega-3 polyunsaturated fatty acids, delivered to the retinal pigment epithelium (RPE) by circulating low-density lipoproteins (LDL), enhance already considerable RPE lysosomal acid lipase activity, providing for more efficient hydrolysis of intralysosomal RPE lipids, an effect that may help prevent development of age-related macular degeneration (ARMD). METHODS: Colorimetric biochemical and histochemical techniques were used to demonstrate RPE acid lipase in situ, in vitro, and after challenge with phagocytic stimuli. Receptor-mediated RPE uptake of fluorescently labeled native, aceto-acetylated, and oxidized LDL was studied in vitro and in vivo. LDL effects on RPE lysosomal enzymes were assessed. Lysosomal enzyme activity was compared in RPE cells from monkeys fed diets rich in fish oil to those from control animals and in cultured RPE cells exposed to sera from these monkeys. RESULTS: RPE acid lipase activity was substantial and comparable to that of mononuclear phagocytes. Acid lipase activity increased significantly following phagocytic challenge with photoreceptor outer segment (POS) membranes. Receptor-mediated RPE uptake of labeled lipoproteins was determined in vitro. Distinctive uptake of labeled lipoproteins occurred in RPE cells and mononuclear phagocytes in vivo. Native LDL enhanced RPE lysosomal enzyme activity. RPE lysosomal enzymes increased significantly in RPE cells from monkeys fed fish oil-rich diets and in cultured RPE cells exposed to their sera. CONCLUSIONS: RPE cells contain substantial acid lipase for efficient metabolism of lipids imbibed by POS phagocytosis and LDL uptake. Diets rich in fish oil-derived omega-3 fatty acids, by enhancing acid lipase, may reduce RPE lipofuscin accumulation, RPE oxidative damage, and the development of ARMD.

Dietary supplementation of N-3 fatty acids and hydroperoxide levels in rat retinas.

Docosahexaenoic acid (DHA) plays an important role in visual and neural development in mammals. In the present study, effect of dietary supplementation with n-3 fatty acids, primarily docosahexaenoic acid (DHA) with high purity, on the fatty acid composition of photoreceptor cells of young rats (fed from 4 weeks) was investigated. DHA in rod outer segment (ROS) membranes was significantly increased in the group of high DHA feeding (9.69% total energy). Other n-3 fatty acids (alpha-linolenic acid (ALA) and eicosapentaenoic acid (EPA)) included in the diets with DHA (0.95%-5.63% total energy) also significantly increased the proportion of DHA compared with the linoleic acid diet groups. However, the proportions of arachidonic acid (ARA) and other long chain n-6 fatty acids (22:4n6 and 22:5n6) were suppressed in these n-3 fatty acids-fed groups. Phospholipid hydroperoxides in ROS membranes were determined using a highly sensitive analytical technique, chemiluminescence-high performance liquid chromatography (CL-HPLC). There was no increasing tendency in the hydroperoxide levels of ROS membranes containing high content of DHA, and phosphatidylethanolamine hydroperoxide (PEOOH) was much lower than phosphatidylcholine hydroperoxide (PCOOH) under normal light conditions, which implies that DHA supplementation does not much affect the peroxidizability of ROS membranes in vivo. But UV irradiation on separated ROS membranes accelerated the formation of phospholipid hydroperoxides in high DHA feeding rats, and PEOOH was produced more efficiently than PCOOH in vitro.

cDNA cloning of the human retinal rod Na-Ca + K exchanger: comparison with a revised bovine sequence.

PURPOSE: To clone the complementary DNA of the human retinal rod Na-Ca + K exchanger. METHODS: A human retinal cDNA library was screened initially with a radiolabeled probe representing the entire bovine rod Na-Ca + K exchanger cDNA and subsequently with probes from polymerase chain reaction fragments of the human retinal rod Na-Ca + K exchanger obtained after the initial screen. Twelve positive clones were used to obtain the entire coding sequence of the human retinal rod Na-Ca + K exchanger. RESULTS: The cDNA of the human retinal rod Na-Ca + K exchanger codes for a protein of 1081 amino acids, which shows 64.3% overall identity with the bovine retinal rod Na-Ca + K exchanger at the amino acid level. The two sets of putative transmembrane-spanning domains and their short connecting loops showed the highest degree of identity (94%-95%), whereas the extracellular loop at the N terminus showed a 59% identity. The large cytosolic loop that bisects the two sets of transmembrane-spanning domains contained two large deletions in the human exchanger; the first deletion contains 18 amino acids, whereas the second deletion involves a series of repeats that are dominated by acidic amino acid residues observed in the bovine, but not in the human, sequence. The authors observed that the bovine sequence contains a ninth repeat in addition to the eight repeats of the published sequence. CONCLUSIONS: The authors cloned the cDNA of the human retinal rod Na-Ca + K exchanger as a first step in examining the possibility that this gene could be the locus of disease-causing mutations.

Structural and functional characterization of a second subfamily member of the calcium-modulated bovine rod outer segment membrane guanylate cyclase, ROS-GC2.

A native bovine calcium-modulated rod outer segment membrane guanylate cyclase (ROS-GC) has been cloned and reconstituted to show its linkage consistent to the process of phototransduction. In the present study, a second form of the membrane guanylate cyclase has been cloned from the bovine retina. This cyclase shares a high sequence identity with ROS-GC, is specifically expressed in the bovine retina, and, like ROS-GC, is modulated in low Ca2+ by a calmodulin-like Ca2+-binding protein, termed GCAP2. For this reason, this cyclase has now been named ROS-GC2 and the previously described ROS-GC as ROS-GC1. The tail end of ROS-GC2 contains a stretch of five amino acids, a structural feature unique to itself. These findings support the existence of a calcium-modulated subfamily of ROS-GC and indicate that ROS-GC2 embodies a five amino acid signature element at its tail end.

Retinal light damage in rats with altered levels of rod outer segment docosahexaenoate.

PURPOSE: To compare retinal light damage in rats with either normal or reduced levels of rod outer segment (ROS) docosahexaenoic acid. METHODS: Weanling male albino rats were maintained in a weak cyclic light environment and fed either a nonpurified control diet or a purified diet deficient in the linolenic acid precursor of docosahexaenoic acid (DHA). Half the rats on the deficient diet were given linseed oil, containing more than 50 mol% linolenic acid, once a week to maintain ROS DHA at near normal levels. Diets and linseed oil supplementation were continued for 7 to 12 weeks. To replenish DHA in their ROS, some 10-week-old rats on the deficient diet were given linseed oil three times a week for up to 3 additional weeks. Groups of animals were killed at various times for ROS fatty acid determinations or were exposed to intense green light using intermittent or hyperthermic light treatments. The extent of retinal light damage was determined biochemically by rhodopsin or photoreceptor cell DNA measurements 2 weeks after exposure and morphologically by light and electron microscopy at various times after light treatment. RESULTS: Rats maintained for 7 to 12 weeks on the linolenic acid-deficient diet had significantly lower levels of DHA and significantly higher levels of n-6 docosapentaenoic acid (22:5n-6) in their ROS than deficient-diet animals supplemented once a week with linseed oil or those fed the nonpurified control diet. As determined by rhodopsin levels and photoreceptor cell DNA measurements, deficient diet rats exhibited protection against retinal damage from either intermittent or hyperthermic light exposure. However, the unsaturated fatty acid content of ROS from all three dietary groups was the same and greater than 60 mol%. In 10 week-old deficient-diet rats given linseed oil three times a week, ROS DHA was unchanged for the first 10 days, whereas 22:5n-6 levels declined by 50%. After 3 weeks of treatment with linseed oil, ROS DHA and 22:5n-6 were nearly the same as in rats supplemented with linseed oil from weaning. The time course of susceptibility to retinal light damage, however, was different. Hyperthermic light damage in rats given linseed oil for only 2 days was the same as for rats always fed the deficient diet. Six days after the start of linseed oil treatment, retinal light damage was the same as in rats given the linseed oil supplement from weaning. Morphologic alterations in ROS of linseed oil-supplemented rats immediately after intermittent light exposure were more extensive than in either the deficient-diet animals or those fed the control diet. The deficient-diet rats also exhibited better preservation of photoreceptor cell nuclei and structure 2 weeks after exposure. CONCLUSIONS: Rats fed a diet deficient in the linolenic acid precursor of DHA are protected against experimental retinal light damage. The relationship between retinal light damage and ROS lipids does not depend on the total unsaturated fatty acid content of ROS; the damage appears to be related to the relative levels of DHA and 22:5n-6.

Relationship between dietary supply of long-chain fatty acids and membrane composition of long- and very long chain essential fatty acids in developing rat photoreceptors.

The present study was designed to determine if dietary supply of long-chain fatty acid (LCFA, C20:4n-6, and/or C22:6n-3), reflecting levels that might be incorporated into infant formulas, influences the fatty acid composition of the visual cell membrane. The rod outer segment (ROS) of the retina was analyzed from rats fed diets varying in the ratio of 18:2n-6 to 18:3n-3 with or without 20:4n-6 [arachidonic acid (AA)] and 22:6n-3 (docosahexaenoic acid) from birth to six weeks of age. The level of very long chain fatty acids (VLCFA, C24-C36) was identified using gas chromatography and gas chromatography-mass spectrometry. In the ROS, the highest relative percent of AA was attained in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of animals fed 1% AA diet, whereas feeding 0.7% docosahexaenoic acid (DHA) diet significantly increased the DHA level in PC, phosphatidylserine, and phosphatidylinositol compared to feeding diets containing AA. VLCFA of n-6 and n-3 up to C36 were found in PC, with the most abundant fatty acids being C32 and C34. In PC, phosphatidylserine and PE, the n-6 tetraenoic VLCFA level was highly increased in animals fed 1% AA compared to other dietary groups. This study suggests that dietary fat containing small amounts of AA or DHA is an important factor influencing membrane fatty acid composition of the visual cell during development.

The renal cGMP-gated cation channel: its molecular structure and physiological role.

Cyclic nucleotide-gated cation channels, which are permeable to monovalent and divalent cations, are expressed in a number of tissues. cDNAs encoding cGMP-gated cation channel subunits have been cloned in retinal rods, cones, olfactory neuroepithelium, pineal gland, aorta, testis, heart, and most recently kidney. Patch clamp studies have identified and characterized cGMP-gated cation channels in the cortical collecting duct (CCD) and inner medullary collecting duct (IMCD). cGMP-gated cation channels in kidney share many biophysical and molecular properties with the retinal rod cGMP-gated channel. However, unlike the retinal rod channel, the cGMP-gated cation channel in kidney is inhibited by cGMP and stimulated by increased calcium levels. In the IMCD the cGMP-gated cation channel mediates electrogenic sodium absorption which is inhibited by ANP via cGMP. Recently, cGMP-gated cation channel poly(A+) RNA has been identified in other nephron segments by RT-PCR and in situ PCR hybridization. Furthermore, cGMP-gated cation channel protein has also been identified in all nephron segments by Western blot analysis. These observations suggest that cGMP-gated cation channels, or closely related gene products, may play an important physiological role in all nephron segments. Hormones that increase intracellular cGMP may regulate sodium, and perhaps calcium, uptake in nephron segments proximal to the IMCD. Increases in cell sodium and calcium may regulate other transport and signaling pathways.