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Results for “Rod Cell Outer Segment”

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PubMed1991

In vivo lectin-binding of photoreceptors and interphotoreceptor matrix in rat.

The in vivo distribution of lectin receptors in the photoreceptors and interphotoreceptor matrix (IPM) was studied by histochemistry and light microscopy. Horseradish peroxidase-conjugated lectins (peanut agglutinin, PNA; Ricinus communis agglutinin-1, RCA-1; or wheat germ agglutinin, WGA) were administered into the subretinal space of living rats, and 1 and 6 hours later eyeballs were enucleated to examine retinal tissues with a light microscope. The surfaces of cone photoreceptor outer segments and inner segments, and/or cone-associated IPM were labeled preferentially with PNA at 1 and 6 hours after its subretinal administration. One hour after subretinal injection, RCA-1 labeling appeared in the distal surface of the outer segments, the basal region of the inner segments, and the surface of the retinal pigment epithelium (RPE). At 6 hours the basal portion of the outer segments showed RCA-1 labeling as well. The distribution of labeling by WGA was uniform throughout the outer segments and/or the IPM domain except for the most distal portion of the outer segments. These observations conform for the most part to previous in vitro experiments, and indicate, together with previously not explicitly described observations, that a spatial heterogeneity in the vertical and horizontal directions exists in the IPM domain.

Animals↗

PubMed1988

Phospholipid species containing long and very long polyenoic fatty acids remain with rhodopsin after hexane extraction of photoreceptor membranes.

About one-fourth the phosphatidylcholines (PCs) from bovine disk photoreceptor membranes contain very long chain (24-36 carbons) polyunsaturated (4, 5, and 6 double bonds) fatty acids of the n-3 and n-6 series (VLCPUFA). Such fatty acids, exclusively occurring in dipolyunsaturated species, are esterified to the sn-1 position of their glycerol backbone, docosahexaenoate being the major fatty acid at sn-2. Chromatographically, such PCs display a weakly polar character relative to other species, ascribable to their exceedingly large number of carbons. After hexane extraction of lyophilized disks, PC is the major component of the fraction of lipids that remains associated with rhodopsin, followed by phosphatidylserine, while a large proportion of the phosphatidylethanolamine is removed. The fatty acid composition of the hexane-removable and protein-bound lipid fractions markedly differs, the latter being enriched in lipid species containing long-chain and very long chain polyenes. This is observed for all lipid classes except free fatty acids. VLCPUFA-containing PCs are the most highly concentrated species in the rhodopsin-associated lipid fraction. The very long chain polyenes these PCs have at sn-1 may account for their resistance to being separated from the protein. It is hypothesized that their unusually long polyenoic fatty acids could be well suited to partially surround alpha-helical segments of rhodopsin.

Animals↗

PubMed1988

Total synthesis and expression of a gene for the alpha-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin).

To facilitate structure-function studies by site-specific mutagenesis, we have synthesized a gene for the alpha-subunit of the bovine rod outer segment (ROS) guanine nucleotide-binding protein (transducin). The gene codes for the native amino acid sequence and contains, by design, 38 unique restriction sites which are uniformly spaced. This enables mutagenesis in any part of the gene by restriction fragment replacement. The gene is 1076 base pairs in length. It was constructed from 44 synthetic oligonucleotides which were joined enzymatically in vitro into four fragments which were cloned. The synthetic transducin gene and cDNA encoding transducin were expressed to similar levels in monkey kidney cells (COS-1) using a vector in which transcription was under the control of the adenovirus major late promoter.

Amino Acid Sequence↗

PubMed1980

An inherited retinopathy in collies. A light and electron microscopic study.

A recessively inherited retinopathy in collies aged 8 to 189 days was studied by light and electron microscopy. The disease is produced when the outer segments of rods and cones fail to develop normally. Retinal pigment epithelial changes found in several litters appeared to form a separate disease entity. We compared the collie retinopathy with other canine models and the collie ectasia syndrome.

Animals↗

PubMed1985

Binding stoichiometry of a fluorescent cGMP analogue to membranes of retinal rod outer segments.

The high-affinity binding of the cGMP analogue 8-(5-thioacetamidofluorescein)-cGMP to rod outer segment membranes depleted of peripherally bound proteins has been defined by equilibrium dialysis (mean +/- SD): membranes contain about one cGMP binding site per 130 rhodopsin molecules; the concentration of free ligand for half saturation is 2.0 +/- 0.6 microM; the apparent Hill coefficient of the bound versus free ligand relationship is 1.7 +/- 0.5; half saturation of the binding sites is sufficient for 85% activation of calcium permeability. A gating mechanism is proposed.

Animals↗

PubMed1988

Nucleotide binding to the rod outer segment rim protein.

The rod photoreceptor outer segment maintains a remarkable morphology. Two of the proteins which have been implicated in the maintenance of this structure are the 240 kDa spectrin-like protein, and the 220 kDa glycoprotein often referred to as the rim protein. We have probed rat rod outer segment proteins with light-activated (azido-labeled) radioactive nucleotides and found a nucleotide binding site(s) on the rim protein which has a preference for guanine nucleotides. Binding to this site is stimulated by the divalent cations zinc, manganese and magnesium, but not calcium. This site is under investigation and may play a role in stabilizing protein structure.

ATP-Binding Cassette Transporters↗

PubMed1987

Light induces a rapid and transient increase in inositol-trisphosphate in toad rod outer segments.

The sub-second time course of changes in the content of [3H]inositol-1,4,5-trisphosphate was determined in rod outer segments from very rapidly frozen Bufo retinas that had been incubated with [3H]inositol. Rod outer segments were cut off frozen specimens with a cryostat microtome and the water soluble extracts were analyzed. The content of [3H]inositol-1,4,5-trisphosphate rose after approximately 250 msec of bright illumination, but returned to the unstimulated level after 1 sec, whether the stimulus remained on or not. That is, there was rapid but transient change in the content of [3H]inositol-1,4,5-trisphosphate after the onset of stimulation.

Animals↗

PubMed1992

Nitroprusside-sensitive and insensitive guanylate cyclases in retinal rod outer segments.

Rod outer segments of retina contain guanylate cyclase activity both in the cytosol and membrane fractions. Though the activity in the cytosol is a small fraction of the total activity, it is highly activated by nitroprusside, a nitric oxide generating agent. The membrane guanylate cyclase on the other hand is unaffected by nitroprusside both before and after solubilization. The effects of nitroprusside or nitric oxide on photoreceptor function should therefore be mediated by the cytosolic and not the membrane guanylate cyclase.

Animals↗

PubMed1984

Location and function of voltage-sensitive conductances in retinal rods of the salamander, Ambystoma tigrinum.

The functional role and spatial location of voltage-sensitive conductances that modify the light-evoked electrical signals were studied in retinal rods of the tiger salamander. An isolated rod was drawn into a suction electrode for recording membrane current and impaled with an intracellular electrode for recording membrane potential and passing current. A bright flash gave a rapid initial hyperpolarization that relaxed to a smaller plateau. Simultaneously the dark current of the outer segment was shut off with the time course of a rounded step function. This characteristic difference between the wave forms demonstrates that the voltage relaxation does not result from reopening of light-sensitive channels. The voltage relaxation in (2) did not require light or interruption of the dark current, as the wave form was duplicated by suddenly switching off a depolarizing current injected during steady saturating light. This is explained if the relaxation depends purely on voltage-sensitive conductances. The voltage response to a dim flash reached its peak value before the current response. The voltage wave form was predicted assuming that the recorded photo-current drove a linear high-pass filter with parameters derived from analysis of the voltage response to injection of a current step. When the intracellular voltage was changed by current injection the slope resistance of the outer segment slowly declined to a lower level, indicating that the outer segment contains a voltage-sensitive conductance. When a current step was injected in bright steady light, the current recorded from the outer segment consisted of a capacity component proportional to dV/dt and a small extracellular leakage current but no detectable ionic current. This supports other evidence indicating that light-sensitive channels comprise the main or exclusive ionic conductance of the outer segment. The behaviour in (5) is explained if the light-sensitive channels themselves are slowly opened by hyperpolarization and closed by depolarization. Analysis of the current-injection experiments suggests that most of the high-pass filtering in a rod results from the action of voltage-sensitive conductances located in the inner segment. Addition of 10 mM-CsCl to the Ringer solution abolished the relaxation in the voltage response to a bright flash but left intact the high-pass filtering of small signals. This would be explained by a selective block of one of two sets of voltage-sensitive channels in the inner segment or by a voltage-sensitive block of one kind of channel.

Ambystoma↗

PubMed1996

Light damage in rod outer segments: the effects of fixation on ultrastructural alterations.

PURPOSE: In this electron microscopical study, we compared effects of chemical fixation versus cryofixation on the ultrastructure of acute rod outer segment alterations in the rat retina. METHODS: The alterations were induced by toxic levels of diffuse white light. Albino rats were exposed to 2000 lux for 30 min. Samples from one eye of each animal were fixed by high pressure freezing and samples from the other eye were fixed by standard glutaraldehyde procedures. RESULTS: Light exposed retina showed major differences in their rod outer segments, inner segments and photoreceptor synaptic regions in chemical fixation. In particular gross vesiculations of outer segment membranes were produced in light exposed experiments. In contrast, in cryo-fixed samples such prominent changes were not observed in outer segment membranes. There was, however, occasional formation of small vesicles and a reduction of the cilium diameter in response to light damage. In the dark adapted control group the morphology of chemically fixed and cryo-fixed photoreceptors was similar. CONCLUSIONS: We conclude, that cryo-fixed samples better represent the living state of the retina, because high pressure freezing is a purely physical method and acts much faster than chemical fixation.

Animals↗

PubMed1994

Expression and mutagenesis of mouse rod photoreceptor cGMP phosphodiesterase.

Using recombinant baculovirus vectors, the three subunits of mouse rod photoreceptor cGMP phosphodiesterase (PDE) (alpha beta gamma 2) have been expressed in insect cells. The recombinant alpha,beta subunits accumulate to 5 mg/liter culture, but most (98%) of the expressed polypeptides are insoluble. In the soluble fraction, individually expressed alpha and beta subunits showed insignificant PDE activity, but coexpression (by coinfection) of alpha beta subunits elevated PDE activity 7-fold and coexpression of alpha beta gamma up to 15-fold. The soluble expressed holoenzyme associated with ROS membranes under isotonic, but not hypotonic, conditions. The Km of the soluble holoenzyme was 11-16 microM both for coexpressed alpha beta subunits and for alpha beta gamma subunits, similar to the Km (6-80 microM) of native PDE. Site-directed mutagenesis of cysteine to serine in the C-terminal CAAX box of both alpha and beta subunits substantially decreased the protein expression level, abolished post-translational isoprenylation, and prevented subunit binding to the rod outer segment (ROS) membranes. The mutant holoenzyme, however, showed a cGMP hydrolytic activity comparable with that of the normal recombinant enzyme. These results suggest that both alpha and beta subunits are required for the formation of a functional enzyme and that isoprenylation of the subunits is essential for membrane association and stability of PDE.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1986

31P-NMR investigation of magnetically oriented rod outer segments. Spectral analysis and identification of individual phospholipids.

A 31P-NMR study of magnetically oriented bovine rod outer segments is presented. We demonstrate that carefully isolated bovine rod outer segments retain the capacity to orient in a magnetic field. Maximal orientation (85-90%) is achieved at field strengths over 4.7 T in the NMR spectrometer. The lineshape of the 'oriented spectra' is totally different from the 'bilayer lineshape' of randomly oriented photoreceptor membranes. The oriented spectra consist of two phospholipid peaks, a major low-field peak (75-80% of the total intensity) near 30 ppm, and a minor high-field peak near - 14 ppm as well as two sharp metabolite peaks around 0 ppm. The phospholipid peaks are a composite of three narrower partially resolved resonances assigned to the individual phospholipid classes phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine. Based on the morphology and magnetic anisotropy of the rod outer segment, the major phospholipid peak is attributed to the flat part of the disk membranes while the phospholipids of the plasma membrane are thought to contribute only to the minor peak. Disk rim phospholipids and non-oriented material contribute to the minor peak and, in addition, contribute some intensity to the middle part of the spectrum. The phospholipid class composition of the major peak is estimated by spectral simulation and is consistent with the phospholipid class composition of rod outer segment membranes. Hence, 31P analysis of oriented rod outer segments resolves the main phospholipids in at least two different membrane pools in the rod outer segment and allows the differential investigation of these pools. Most of the mobile phosphate metabolite intensity resides in the Pi peak at 3.5 ppm. A slight shift in the Pi resonance position indicates a 0.2 pH unit acidification upon illumination of rhodopsin. The absence of detectable nucleotide resonances, when compared with chemical analysis, indicates that the majority of the nucleotide population present is rather immobile and probably bound to the membranes.

Animals↗

PubMed2001

Retina of Bouton's skink (Reptilia, Scincidae): visual cells, fovea, and ecological constraints.

Bouton's skink, Cryptoblepharus boutonii africanus, is a small, diurnal lizard living on outcrops along the coast of East Africa under high ambient light intensities. It is characterized by relatively large eyes (maximal diameter about 2 mm), with immovable eyelids forming a transparent spectacle and with a virtually constant pupil diameter. The single fovea in the central retina is well developed, with a clearly defined pit, which is relatively deep but not funnel-shaped. The foveal pit is not devoid of the outer nuclear and outer plexiform layers; only the main part of the inner nuclear layer is displaced laterally, resulting in a pit with gradual sloping towards its edges. Thus, the fovea appears to be concaviclivate, as in the eyes of lacertids, varanids, and gekkonids. The central position of the foveae in these laterally placed scincid eyes corresponds with monocular fixation, e.g., of detected prey. C. boutonii has a pure-cone retina containing single and double visual cells. The latter consist of two cells of unequal sizes. Yellowish oil droplets are present in single cones and the minor members of the double cones in all retinal regions. The visual cells of the different retinal regions do not differ in the ultrastructure of their components but differ considerably in size. The outer segments of the foveal cones are twice as long as those of the peripheral cones. Except for the pedicles, the diameters of the components of the visual cells decrease towards the fovea, resulting in an increase in visual acuity.

Animals↗

PubMed1981

[Photo-induced release of calcium ions frog retina photoreceptor membranes in media of different ionic composition].

Two methods: labelled Ca45 and Ca++-selective electrode served to assess the Ca++ release during illumination of the rod outer segments in the frog retina. The maximal release (8--10 M Ca++/M bleached rhodopsin) occurred in monovalent cations--free medium (100 MM tris--HCl, 5 mM MgCl2, 5 mM ATP, pH--7.5), when at least 50% of visual pigment was bleached. The addition of Na+ or K+ toincubation medium decreased the photoinduced release to 0.06--0.142 M Ca++/M bleached rhodopsin or completely suppressed it. The role of Ca++ as a messenger during the excitation of photoreceptor is discussed.

Adenosine Triphosphate↗

PubMed1990

Identification of a C-terminal protein carboxyl methyltransferase in rat liver membranes utilizing a synthetic farnesyl cysteine-containing peptide substrate.

Polypeptides synthesized in eucaryotic cells with a C-terminal -Cys-Xaa-Xaa-Xaa (-CXXX) sequence are candidates for post-translational modifications that include the removal of the last 3 amino acids and the lipidation and methyl esterification of the cysteinyl residue. To characterize the methylation reaction in vitro, the peptide Leu-Ala-Arg-Tyr-Lys-Cys (LARYKC) and its S-isoprenylated and S-alkylated derivatives were synthesized and assayed as methyl-accepting substrates with subcellular fractions of rat tissues including liver microsomal membranes. While little or no peptide-specific methyltransferase activity was detected in the latter preparation using the unmodified hexapeptide, the C10, C15, and C20 isoprenylated derivatives were substrates with Km values of 389 microM for S-geranyl-LARYKC, 2.2 microM for S-farnesyl-LARYKC, and approximately 10.9 microM for S-geranylgeranyl-LARYKC. The methyl-acceptor activities of a variety of n-alkyl S-derivatives of LARYKC (C8, C10, C13, C15) were also tested; all of these compounds were poorer substrates than the S-geranyl derivative. This enzyme activity uses S-adenosyl-L-methionine as the methyl donor (Km = 2.1 microM) and can be inhibited by S-adenosylhomocysteine (Ki = 9.2 microM), a product of the methylation reaction. The S-farnesyl-LARYKC peptide can inhibit the carboxyl methylation of bovine retinal rod outer segment membrane proteins that was previously shown to occur at the alpha-carboxyl group of C-terminal cysteine residues, demonstrating that the same enzyme can methylate both peptides and proteins. These results suggest that the methyl esterification of proteins containing a C-terminal -CXXX sequence requires not only the removal of the 3 terminal amino acids, but the isoprenylation of the sulfhydryl group as well.

Amino Acid Sequence↗

PubMed1997

Biochemical characterization of cell specific enzymes in light-exposed rat retinas: oxidative loss of all-trans retinol dehydrogenase activity.

PURPOSE: To determine the effect of acute, intense, visible light on the activities of all-trans retinol dehydrogenase (t-RDH) and glutamine synthetase (GS), two oxidatively sensitive enzymes located in the photoreceptors and Müller cells, respectively. METHODS: Male albino rats, previously maintained in a weak cyclic light- or dark-rearing environment, were exposed to intense light (490-580 nm) for as long as 24 hrs. One-half of the experimental animals were pre-treated with the antioxidant 1,3-dimethylthiourea (DMTU), at 500 mg/kg, i.p., 24 hrs before and just before light exposure. Upon sacrifice, retinas were excised for the determination of t-RDH and GS activity, or for the preparation of rod outer segments (ROS). Other light-exposed rats were maintained in darkness for 2 weeks before sacrifice, for rhodopsin determinations. Retinal homogenates were also treated in vitro under oxidizing conditions to compare enzymatic inactivation with the in vivo effects of light exposure. RESULTS: In cyclic light-reared rats 24 hr light exposures resulted in a significant loss of t-RDH activity in retinal homogenates and in isolated ROS. In both the retina and ROS, pretreatment of the animals with DMTU completely prevented the loss of t-RDH activity. As measured by rhodopsin levels 2 weeks after light exposure, DMTU-treated rats exhibited no loss of photoreceptor cells, whereas those not given the antioxidant lost over 50% of their photoreceptors. Retinal GS activity was unchanged by 24 hr intense light exposures. In dark-reared rats 4 hr light exposures did not alter retinal t-RDH or GS activity, despite the loss or approximately 70% of the rhodopsin content or the eye, measured 2 weeks later. When 4 hr light-exposed rats were held in darkness for an additional 20 hrs, a significant loss of retinal t-RDH occurred, but no change in GS activity was measured. In these rats DMTU treatment also prevented the loss of t-RDH activity. In contrast to the lack of an in vivo light effect on retinal GS, oxidation in vitro completely inactivated the enzyme after only 1 hr. CONCLUSIONS: The light-induced loss of t-RDH in both cyclic light- and dark-reared rats is an oxidative and time dependent process that is not strictly photochemical in nature. The loss of rhodopsin and t-RDH activity, but not GS activity, following intense light exposure are manifestations of light's effect on photoreceptor cells without a comparable effect in the adjacent retinal Müller cells. Additional work will be needed to understand the differences in light damage susceptibility between retinal photoreceptors and glial cells and between cyclic light- and dark-reared rats.

Alcohol Oxidoreductases↗

PubMed1989

Lipofuscin-like substances accumulate rapidly in brain, retina and internal organs with cysteine protease inhibition.

The protease inhibitor, leupeptin, has been previously shown to cause an accumulation of lysosomally associated intracytoplasmic dense bodies resembling lipofuscin when administered intraventricularly to the brains of young rats (Ivy et al., Science, 226:985, 1984). These findings support the idea that lipofuscin formation during normal aging involves perturbed proteolytic degradation. In the current study, we delineate more precisely the proteolytic mechanisms in question and examine the effects of protease inhibition on other tissues and species. Four sets of experiments were done. In the first, rats were administered leupeptin (L; an inhibitor of cysteine and some serine proteases), E-64C (a cysteine protease inhibitor), chloroquine (C; a lysosomal enzyme inhibitor), aprotinin (A; a serine protease inhibitor) or physiological saline (S) into the lateral ventricle of the brain at a constant rate for two weeks using an osmotic mini-pump. In the second set, beagle dogs were administered L or S intraventricularly in a similar fashion. In the third set, young rats received intraocular injections of L, E64C, C, A or S from 1 to 9 days at 24 hr intervals. In the fourth set, young rats and mice were administered L, E-64C or S intraperitoneally via an osmotic mini-pump for 2 or more weeks. The tissue of all animals was processed for light and electron microscopy as well as for fluorescence analysis. In rat brain cells (especially hippocampus and cerebellum), substances which by morphological, histochemical and fluorescence emission criteria resemble lipofuscin, accumulated following L, C or E64C, but not A or S treatment. A similar buildup of lipofuscin-like substance occurred in canine brain following L but not S infusion. In retinal pigment epithelial cells, undigested rod outer segment discs accumulated after L, C or E-64C and possibly A, but not after S injections. With increasing survival times, the accumulated substances became morphologically more similar to lipofuscin. Cells of liver (both hepatocytes and non-parenchymal cells) and kidney (mainly renal tubule cells) also accumulated substances resembling lipofuscin in response to L but not S infusion; other internal organs were differentially affected. These results demonstrate 1) that specific inhibition of cysteine proteases is sufficient to cause accumulation of lipofuscin-like substances in brain and retina and 2) that several species and organ systems are similarly affected by protease inhibition. Taken together, these findings are consistent with the idea that defective or decreased proteolysis by lysosomal cysteine proteases is responsible for lipofuscin accumulation during normal aging.

Aging↗

PubMed1995

Ultrastructural features of retinal dystrophy in mutant vitiligo mice.

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

Animals↗