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PubMed1985

Calcium content and calcium exchange in dark-adapted toad rods.

We have used laser-activated micro mass analysis (l.a.m.m.a.) and energy-dispersive X-ray analysis (e.d.x.) to measure Ca content and Ca movements in 'red' rod photoreceptors in the dark-adapted retina of the toad, Bufo marinus. Measurements with both l.a.m.m.a. and e.d.x. show that intact rod outer segments contain 4-5 mmol total Ca/l wet tissue volume, or 1-2 Ca per rhodopsin. We could detect no significant variation in the total Ca as a function of distance across or up and down the outer segment. In the inner segment, Ca could be detected only within the mitochondria-rich ellipsoid body, where the total Ca concentration was of the order of 100-400 mumol/l wet tissue volume. To measure the exchange of Ca in outer segments from intact photoreceptors, we exposed the dark-adapted retina to Ringer containing the stable isotope 44Ca. Since l.a.m.m.a. can measure separately the concentrations of each of the isotopes of the elements, and since native rods contain almost exclusively 40Ca, the increase in 44Ca and decrease in 40Ca could be used as a measure of Ca influx and efflux. Ca exchange in intact rod outer segments in darkness is very slow. The rate of accumulation of 44Ca was only 10(5) Ca/rod.s, or about 10% of the total outer segment Ca/h. This slow rate of exchange is apparently not the result of restricted movement of Ca across the plasma membrane. Ca exchange was also measured in outer segments which were either partially or entirely detached from the rest of the photoreceptor. In broken-off outer segments, Ca exchange is faster than in the intact organelles, and in 1 h, half of the 44Ca exchanges for 40Ca. When the retina was incubated in Ringer for which all of the Na was substituted with Li or choline, there was an increase in the rate of 44Ca accumulation in intact outer segments, probably due to an inhibition of Na-Ca counter transport across the plasma membrane. Our measurements indicate that the great majority of the Ca in the rod appears to be inaccessible to exchange under physiological conditions, probably because it is sequestered within the disks which in intact rods appear to be nearly impermeable to Ca in darkness.

Animals↗

PubMed1983

Simple sugars inhibit rod outer segment disc shedding by the frog retina.

To investigate the hypothesis that sugar molecules might act as markers for ROS disc phagocytosis, frog eyecups were incubated in a normally permissive medium for ROS disc shedding with and without added simple sugars. L-fucose, alpha-methyl-D-mannopyranoside and D-mannose all significantly reduced the numbers of packets of ROS discs found in the retinal pigment epithelium. D-fucose, L-mannose, D-fructose, D-galactose, D-glucose and sucrose were without significant effect at the same concentration. Ultrastructural examination of the retinas indicates that the sugars were effective on the disc shedding process rather than on phagocytosis of already shed disc packets.

Animals↗

PubMed1999

Association of the tyrosine phosphatase SHP-2 with transducin-alpha and a 97-kDa tyrosine-phosphorylated protein in photoreceptor rod outer segments.

Increasing evidence indicates that tyrosine phosphorylation, controlled by the concerted action of tyrosine kinases and protein tyrosine phosphatases (PTPs), plays important roles in retinal photoreceptor rod outer segments (ROS). We characterized PTP activity in isolated bovine ROS that is significantly inhibited by orthovanadate. Incubating ROS in the presence of exogenous Mg2+, ATP, and orthovanadate dramatically enhanced the tyrosine phosphorylation of several endogenous proteins. SHP-2, a PTP with two SH2 domains, was identified in ROS by immunoblot analysis and was found to associate with ROS membranes. Immunocytochemistry showed localization of SHP-2 in photoreceptor outer segments and possibly in the outer plexiform, inner nuclear, and inner plexiform cell layers of the retina as well. SHP-2 associated with transducin-alpha and a 97-kDa tyrosine-phosphorylated protein in ROS, suggesting the formation of a multimeric signaling complex. Based on its association with transducin-alpha and a 97-kDa protein, SHP-2 may regulate the tyrosine phosphorylation of endogenous proteins, including transducin-alpha, and may play a significant role in a novel signaling pathway in photoreceptors.

Animals↗

PubMed1992

Rod outer segments are designed for optimum photon detection.

The proposal that rod outer segment length is optimal with respect to photon absorption and noise control is extended and tested in a number of species. We find good agreement with our optimality criterion in duplex retinae where rods act as detectors of one or a few photons, but not in all rod retinae nor in those which are exposed to significant photic environmental noise.

Absorption↗

PubMed1981

Distinct states of lipid mobility in bovine rod outer segment membranes. Resolution of spin label results.

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0 degrees C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24 degrees C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0 degrees C and a considerable temperature dependence. This component which is not resolved at high temperatures (24--35 degrees C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62--64 G at 0 degrees C, with very little temperature dependence (61--62 G at 35 degrees C), and is attributed to protein aggregation.

Animals↗

PubMed1981

Activation of phosphodiesterase in frog rod outer segment by an intermediate of rhodopsin photolysis I.

Guanosine 3',5'-cyclic monophosphate phosphodiesterase (EC 3.1.4.1) in frog rod outer segment prepared by a sucrose stepwise density gradient method was activated by light in the presence of GTP. Rhodopsin in rod outer segment was solubilized with sucrose laurylmonoester and then purified by concanavalin A-Sepharose column. Addition of photo-bleached preparation of the purified rhodopsin to the crude rod outer segment, which had been prepared by 43% (w/w) sucrose floatation, caused the activation of phosphodiesterase in the dark, while each component of the photo-product eluted from the column (all-trans retinal and opsin) did not. Regenerated rhodopsin prepared from 11-cis retinal and purified opsin activated phosphodiesterase when it was bleached. From these facts it is suggested that an intermediate or a process of photolysis of rhodopsin causes activation of phosphodiesterase.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2006

Defective development of photoreceptor membranes in a mouse model of recessive retinal degeneration.

Retinal neurodegeneration occurs in several inherited diseases. Some of the most severe disease alleles involve mutations at the C-terminus of rhodopsin, but in no case is the pathogenic mechanism leading to cell death well understood. We have examined a mouse model of recessive retinal degeneration caused by a knock-in of a human rhodopsin-EGFP fusion gene (hrhoG/hrhoG) at the rhodopsin locus. Whereas heterozygous mutant mice were indistinguishable from control mice, homozygous mutant mice had retinal degeneration. We hypothesized that degeneration might be due to aberrant rhodopsin signaling; however, inhibiting signaling by rearing mice in total darkness had no effect on the rate of degeneration. Using confocal and electron microscopy, we identified the fundamental defect as failed biogenesis of disk membranes, which is observed at the earliest stages of outer segment development. These results reveal that in addition to its role in transport and sorting of rhodopsin to disk membranes, rhodopsin is also essential for formation of disks.

Animals↗

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↗

PubMed2004

Effects of extremely low frequency electromagnetic fields on the adenylate kinase activity of rod outer segment of bovine retina.

Extremely low frequency electromagnetic fields (ELF-EMFs) of 75 Hz with amplitudes above a threshold of about 125 microT have a dramatic effect on the adenylate kinase (AK) activity of the rod outer segment (ROS) membranes. In fact, the ATP production by ROS membranes or by purified disk membranes placed in the field decreased by approximately 54%. The decrease in enzymatic activity was independent of the time of exposure to the field and was completely reversible. When disk membranes were solubilized with Triton or a soluble isoform of AK was used, negligible effects of the field were obtained on the enzymatic activity, suggesting that the membrane has an important role in determining the conditions for the enzyme inactivation.

Adenosine Triphosphate↗

PubMed1982

Intraretinal recordings of slow electrical responses to steady illumination in monkey: isolation of receptor responses and the origin of the light peak.

The onset of steady illumination of the mammalian retina initiates a series of complex electrical responses. Corneal recordings in man show a relatively fast ERG, followed by a much slower "light response": a "light peak" followed by a series of damped slow oscillations that may last for 1 or 2 hr. The latter oscillations are a prominent feature of the EOG. We recorded the response to steady illumination with micro electrodes in the intact rhesus monkey eye. The intraretinal "light response" was analyzed by simultaneous recordings at different depths, using a bipolar microelectrode. We found that a steady photoreceptor component can be isolated by fractional recording across the cone outer segment layer in the fovea. Further, through simultaneous recordings at the retinal and choroidal sides of the retinal pigment epithelium, we found that this structure is most probably the generating site of the "light peak" and subsequent oscillations of the standing potential.

Animals↗

PubMed1980

Spontaneous quantal events induced in toad rods by pigment bleaching.

Barlow proposed that absolute visual threshold is limited by photon-like noise events in the rod photoreceptors, and he later extended the idea to explain the elevation of threshold following bright bleaching lights in terms of increased noise in the photoreceptors. Rushton, on the other hand, has proposed that the threshold elevation during dark adaptation involves changes not within the photoreceptors themselves but rather in the gain of a subsequent 'pool'. I report here measurements of outer segment current in individual rod photoreceptors which demonstrate that spontaneous fluctuations occur at a greatly increased rate following bleaches of around 1%, and that these fluctuations have the form expected for random occurrences of the single photon events. This is consistent with Barlow's ideas but does not indicate whether a gain change subsequent to the receptors also occurs.

Action Potentials↗

PubMed1988

The concentration of cytosolic free calcium in vertebrate rod outer segments measured with fura-2.

The use of fluorescent indicators such as fura-2 (Grynkiewicz et al., 1985) to measure the cytosolic free calcium activity in retinal rods is complicated by the rods' sensitivity both to the fluorescence and to the light that excites it. By stimulating fluorescence from large numbers of rods in whole, loaded retinas and averaging repeated measurements, however, we have been able to monitor changes in free [Ca2+]i during exposure to nonsaturating lights under physiological conditions. Retinas, isolated from the bullfrog Rana catesbeiana, were loaded with fura-2 by incubation and mounted, receptor-side up, in a perfusion chamber placed on the stage of a specially designed apparatus. A step of light delivered from above, whose wavelength alternated between 340 and 380 nm every 110 msec, excited fluorescence from 24 mm2 of retina and evoked a light response (the aspartate-isolated pIII component of the electroretinogram--ERG). By comparing the fluorescence intensities excited by the 2 wavelengths (corrected for background and dark-noise), the free [Ca2+]i of the rod outer segment was determined. In darkness, the [Ca2+]i of the outer segment was found to be approximately 220 nM. A bright light caused it to fall exponentially to approximately 140 nM, with a time constant of approximately 1.6 sec. The value of [Ca2+]i at the onset of illumination was independent of stimulus intensity over a 2 log-unit range, and in all cases the fall was monotonic. After terminating the illumination, [Ca2+]i rose again to its time-zero value.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗

PubMed2004

Structural, biochemical, and functional characterization of the calcium sensor neurocalcin delta in the inner retinal neurons and its linkage with the rod outer segment membrane guanylate cyclase transduction system.

This study documents the detailed biochemical, structural, and functional identity of a novel Ca(2+)-modulated membrane guanylate cyclase transduction system in the inner retinal neurons. The guanylate cyclase is the previously characterized ROS-GC1 from the photoreceptor outer segments (PROS), and its new modulator is neurocalcin delta. At the membrane, the myristoylated form of neurocalcin delta senses submicromolar increments in free Ca(2+), binds to its specific ROS-GC1 domain, and stimulates the cyclase. Neurocalcin delta is not present in PROS, indicating the absence of the pathway in the outer segments and the dissociation of its linkage with phototransduction. Thus, the pathway is linked specifically with the visual transduction machinery in the secondary neurons of the retina. With the inclusion of this pathway, the findings broaden the understanding of the existing mechanisms showing how ROS-GC1 is able to receive and transduce diverse Ca(2+) signals into the cell-specific generation of second-messenger cyclic GMP in the retinal neurons.

Amacrine Cells↗

PubMed1987

Acylation of disc membrane rhodopsin may be nonenzymatic.

Bovine retinal rod outer segments (ROS) support the incorporation of [3H]palmitate into rhodopsin. [14C] Palmitoyl-CoA serves as the donor with an apparent Km of 40 microM. Solubilization of ROS in the detergent, Emulphogene, results in increased incorporation of label into rhodopsin. A further increase is found when ConA-Sepharose-purified rhodopsin is used as the source of both "enzyme" and acceptor. Failure to separate enzyme from acceptor suggested the possibility of a nonenzymatic reaction. This was confirmed when boiled rhodopsin was found to support the reaction. However, the acylation of rhodopsin is not an artifact since analysis of purified native rhodopsin reveals the presence of covalently bound palmitate and we showed that whole bovine retinas incubated with [3H] palmitate incorporated the fatty acid into rhodopsin (O'Brien, P.J., and Zatz, M. (1984) J. Biol. Chem. 259, 5054-5057). Furthermore, in vivo experiments with rat retinas have revealed that opsin is acylated both in the rod inner and outer segments (St. Jules, R. S., and O'Brien, P.J. (1986) Exp. Eye Res. 43, 929-940). Incubation of labeled rhodopsin with mercaptoethanol resulted in release of the labeled palmitate indicating the presence of a thioester bond. This also illustrates the ease with which a thioester, such as palmitoyl cysteine or palmitoyl-CoA, can transfer the fatty acyl group to a free thiol, such as cysteine or mercaptoethanol.

Acylation↗

PubMed1985

Effects of fluoride on retinal rod outer segment cGMP phosphodiesterase and G-protein.

The effects of fluoride on ROS phosphodiesterase and G-protein have been studied using membrane-free extracts. When G-protein was present NaF, at millimolar concentrations, stimulated PDE activity however, in a G-protein free extract, cGMP hydrolysis was inhibited by high fluoride concentrations. Fluoride was also found to profoundly inhibit the ability of G-protein to bind a GTP analogue, GTP gamma S, both in the presence and absence of rhodopsin. Aluminium greatly modified these effects of fluoride on PDE and G-protein. The possibility that fluoride activates PDE through its effect on G-protein is discussed.

3',5'-Cyclic-GMP Phosphodiesterases↗

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↗

PubMed1986

Rod photoreceptor cells dissociated from mature mice retinas.

Intact rod photoreceptors were dissociated from pronase-treated whole retinas of adult mice by repeated passage through a plastic pipette tip. Hemocytometer counts of the cell suspensions indicate that, during a series of ten dissociation steps, a total of about 1-2 million intact photoreceptor cells are dissociated from one adult mouse retina, with less than 5% contamination from Müller cells and neurons of the inner retina. Visual cells with rod outer segments (ROS) and synaptic terminals are released in each step, but they occur in the greatest number during the sixth to ninth steps; detached ROS are released most frequently in the early steps, and neurons of the inner retinal layers appear in the later steps of dissociation. Nuclei are found in each step. Cell intactness was estimated by Trypan blue and Erythrosin B exclusion and by microscopic analysis using differential interference optics or scanning electron microscopy. The cells bind lectins (concanavalin A, Ricinis communis, and wheat germ agglutinin but not peanut agglutinin), displaying surface topography like that observed in situ. The metabolic capacity of dissociated cells was assessed by measuring the utilization of 32P inorganic phosphate for the synthesis of phospholipids and for the light-dependent phosphorylation of rhodopsin. Mature photoreceptor cells were estimated to contain, on average, 6.4 X 10(-12) g DNA, 2.3 X 10(-12) g RNA and 42-64 X 10(-12) g protein. The dissociation procedure provides a population of photoreceptor cells that appears suitable for microscopic, electrophysiological, and biochemical analysis.

Animals↗

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↗