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PubMedDate not supplied

cGMP-dependent cation channel of retinal rod outer segments.

Light-modulated cytoplasmic cGMP simultaneously controls plasma membrane Na+ conductance in visual excitation and Ca2+ entry into rods by direct interaction with the cation channel. Cytoplasmic Ca2+ in turn may set operating points and contribute to the dynamics of several enzymes that regulate cGMP levels in the dark, recovery from excitation and receptor adaptation or down regulation. Similar channels may couple electrical activity to internal nucleotide metabolism in other tissues. We here report the identification, partial purification and behaviour after reconstitution of a protein of relative molecular mass 39,000 (Mr 39K) present in both disk and plasma membranes from bovine rod outer segments that mediates these cGMP-dependent cation fluxes. Its cGMP agonist specificity, kinetic cooperativity, ionic selectivity, membrane density and other features closely match the properties of the visual cGMP-dependent conductance inferred from electrophysiological measurements.

Animals↗

PubMed1992

The ultrastructure of transplanted rabbit retinal epithelium.

Retinal epithelial cells from pigmented rabbits have been cultured and radiolabelled during division with 3H-thymidine and transplanted to the subretinal space of albino rabbits using a pars plana transvitreal approach. The host rabbits were maintained on cyclosporine immunosuppression after transplantation surgery. The transplant cells survive and maintain a morphologically normal appearance for at least 5 months. The pigmented epithelial cells retain their pigmentation and become integrated with the host retinal epithelium forming junctional complexes with both the host and other transplant cells. The transplant cells contact host photoreceptors with their apical processes and phagocytize outer segments. There appears to be no significant cell division among the transplant cells.

Albinism↗

PubMed1981

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

Frog (Rana catesbeiana) rod outer segment membrane contains cyclic GMP phosphodiesterase (EC 3.1.4.1). Irradiation of dark-adapted rod outer segment membrane increased the enzyme activity by 5-20-fold in the presence of GTP. The phosphodiesterase in rod outer segment membrane is also activated by mixing a photo-product of 11-cis (regenerated), 9-cis or 7-cis rhodopsin which is stable at 0 degrees C. However, neither opsin in the membrane nor all-trans retinal activates the enzyme. The phosphodiesterase in rod outer segment membrane is also activated by irradiation at -4 degrees C. Thus, we conclude that the phosphodiesterase in activated by a common photolysis intermediate of these rhodopsin isomers, perhaps before metarhodopsin II decays.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1981

RCS rat macrophages exhibit normal ROS phagocytosis.

Peritoneal macrophages from RCS rats exhibit phagocytic capability equal to that found in macrophages from normal strains of rat. The similarity in phagocytic activity was found with rat or bovine rod outer segments as well as with dystrophic retinal debris. Thus the genetic defect in RCS pigment epithelium is not expressed in the macrophages, which are available to clear the debris layer that accumulates in dystrophic RCS retinas.

Animals↗

PubMed1958

The effect of illumination on the electrical conductance of rhodopsin solutions.

An apparatus was constructed in order to record continuously and simultaneously changes in extinction and electrical conductance of rhodopsin solutions. With this apparatus, changes in electrical conductance on exposing rhodopsin to light were investigated. On illumination solutions of rhodopsin revealed a conductance change so long as they preserved their photosensitivity. The conductance change begins almost immediately upon illumination and is almost proportional to the amount of rhodopsin decomposed, continuing until rhodopsin is converted to indicator yellow. Near pH 7 the conductance is apt to increase slightly, while it decreases considerably outside the range of pH 6-9, being accompanied by a pH change towards neutrality. The conductance change is regarded as an essential property of rhodopsin, because it occurs in aqueous suspension as well as in digitonin solution; it may be caused by hydrogen or hydroxyl ions and some other conductive substances. It is also noteworthy that the petroleum ether-soluble component of the rod outer segments-presumably the lipide-tends to increase the conductance change. In suspensions of rod outer segments and retinal homogenates, the conductance increases on illumination irrespective of pH: this may be due to secondary reactions following the photic reaction of rhodopsin. We shall discuss the significance of the conductance change in relation to the initiation of visual excitation.

Electric Conductivity↗

PubMed1986

Ultrastructural localization of retinal S antigen in the human retina.

Retinal S antigen, a uveitogenic protein implicated in several forms of clinical and experimental uveoretinitis, has been localized by immunoelectron microscopy in the human photoreceptor outer segment using a protein A-gold technique and anti-bovine retinal S antigen serum. The protein was predominantly located to the proximal portion of the rod outer segment and appeared to be distributed on the disc membrane but not on the plasma membrane. No immunoreactive product was detected in cone outer segments or in any other retinal structure. These findings are discussed in relation to the physiological function of S antigen and its role in initiation of posterior uveitis.

Antigens↗

PubMed1988

Phospholipase C activity and substrate specificity in frog photoreceptors.

The photoreceptor, like many other cells, undergoes receptor-mediated breakdown of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. The lack of phosphatidylinositol (PdtIns) breakdown during receptor stimulation suggests the existence of a phospholipase C specific for polyphosphoinositides. Phospholipase C activity in frog rod outer segments was assayed with several substrates. The activity was selective for the polyphosphoinositides, phosphatidylinositol 4-phosphate [PtdIns(4)P] and PtdIns(4,5)P2. PtdIns was hydrolysed at 2% of the rate of hydrolysis of PtdIns(4,5)P2. No activity was detected when phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine were used as substrates. The enzymatic activity was optimal at neutral pH. These findings suggest, but do not prove, that this activity might contain the light-regulated phospholipase C of the photoreceptor.

Animals↗

PubMed1999

Neither functional rod photoreceptors nor rod or cone outer segments are required for the photic inhibition of pineal melatonin.

Pineal melatonin production is rapidly suppressed by light. In mammals, the photoreceptors mediating this response are ocular; however, definitive information regarding their nature and precise location is absent. In an attempt to define these photoreceptors, we examined the sensitivity of pineal melatonin production to inhibition by controlled irradiance monochromatic green light (lambda max 509 nm) in C3H mice bearing either of two mutations affecting the retina: retinal degeneration (rd), a disruption of rod phototransduction, and retinal degeneration slow (rds), an ablation of photoreceptor outer segments. Diurnal profiles of pineal melatonin content were similar in both mutant genotypes and in wild-type mice; melatonin peaked between 3-5 h before lights on. All three genotypes exhibited irradiance dependent inhibition of pineal melatonin content; 2.6 x 10(-2) microwatts/cm2 509 nm light induced complete suppression in all three genotypes, whereas lower irradiances were ineffective in all cases. Bilateral enucleation abolished responses even to 6 microwatts/cm2 509 nm light. These results demonstrate that the process of irradiance detection for pineal melatonin inhibition is buffered against considerable loss of photoreceptive capacity and that neither rod photoreceptors nor rod or cone outer segments are required for mediating this response in mice.

Animals↗

PubMed1994

Parallel regulation of fetal gene expression in different photoreceptor cell types.

Transcriptional induction for multiple bovine rod-specific photoreceptor genes has been shown to coincide with the first emergence of rod outer segments (two-thirds gestation) suggesting a coordinate regulation of these genes. Since cone visual transduction proteins are encoded for by distinct genes and mammalian cone cell genesis has been found to precede rod cell genesis it is important to determine when induction of photoreceptor-specific gene expression occurs in cone cells and how the timing of this event compares to that in rods. RNase protection assays specific for each cell type-specific gene were used to compare mRNA levels for bovine rod and blue cone gamma subunits of cGMP phosphodiesterase and rhodopsin and red cone opsin from before transcriptional induction (4 months gestation) to the adult. Both pairs of rod and cone visual transduction mRNAs exhibit indistinguishable transcriptional induction points at around 6 months of gestation. Before this time, transcripts of all four genes are present at low, but detectable levels. Thus, although rod and cone cells are distinct cell types and cone cells may be committed well before rod cells early in development, during the final trimester when elaboration of outer segments begins, there appears to be a coordinated regulation of both rod- and cone-specific visual transduction genes.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1995

IP3 generation increases rod outer segment phagocytosis by cultured Royal College of Surgeons retinal pigment epithelium.

PURPOSE: To measure outer segment phagocytosis in cultures of Royal College of Surgeons (RCS) rat retinal pigment epithelium (RPE) that have been treated with carbachol. Carbachol treatment of RCS RPE results in an increase in the second messenger inositol triphosphate, which mimics that observed in normal RPE after interaction with rod outer segments (ROS). METHODS: Cultures of RCS RPE were phagocytically challenged with isolated rat ROS for 2 hours. Carbachol (1 mM) was added to some cultures to stimulate inositol triphosphate synthesis, and incubation continued for 30 minutes at 37 degrees C. Inositol triphosphate concentration was measured by radioreceptor assay. Bound and ingested outer segments were quantified by double immunofluorescent staining. Ingestion of outer segment membranes was confirmed by electron microscopy and immunogold staining. RESULTS: Carbachol treatment was associated with a rapid and temporary increase in inositol triphosphate levels. Royal College of Surgeons rat RPE phagocytically challenged with outer segments and treated with carbachol showed significantly higher ingestion (34%) compared to untreated RCS RPE (9%) (P < 0.05). CONCLUSIONS: Exposure of cultured RCS RPE to carbachol increases the intracellular concentration of inositol triphosphate and enhances phagocytosis of bound ROS. These results support the hypothesis that the phagocytic defect in RCS RPE is related to an abnormality in the generation of inositol triphosphate as a second messenger after outer segment recognition and binding.

Animals↗

PubMed1987

The number, depth and elongation of disc incisures in the retinal rod of Rana catesbeiana.

The disc incisures in the bullfrog retinal rod were examined by electron microscopy. The number of disc incisures is proportional to the cross-sectional perimeter of the outer segment. While the diameter of the red rod outer segments varies with their location along the vertical meridian of the retina, the incisure number also changes similarly. It is estimated by regression analysis that a single disc lobule, bordered by two adjacent incisures, has on the average about 1 micron of arc-like rim edge. When red and green rod discs are compared at their middle outer segments in the ventro-central retina, the disc incisures are 1.8 times more numerous and 1.5 times deeper in the red rod (mean incisure number, 27; mean length, 2.7 microns) than in the green rod (15; 1.8 microns). These differences result in the total disc edge of the red rod (mean, 178 microns) being two times longer than that of the green rod (85 microns). The disc incisures emerge at the basal end of the outer segment and on the average become linearly longer toward the middle outer segment. During displacement of discs over the distance of 2.5 microns, which covers about 90 discs, the incisures reach more than 90% of their maximum length of 2.7 micron in the middle outer segment. In addition, the process of incisure elongation has a kind of fluctuation in that the incisure frequently shows a sharp decrease and increase in length accompanied by a change in course in the disc surface.

Animals↗

PubMed1987

[Methylation of 3',5'-cGMP phosphodiesterase in retinal photoreceptor membranes].

It was found that the level of methylation of phosphodiesterase (EC 3.1.4.17) and of proteins with Mr 61000 and 21000-26000 Da in isolated preparations of bovine retinal outer segments was sharply increased in the presence of cGMP, but not with cAMP. Preparations of the outer segment phosphodiesterase protein inhibitor, which inhibits the cyclic nucleotide hydrolysis, significantly decreased the methylation level. The degree of the enzyme methylation depended on the functional state of the outer segments: in light-induced preparations it was higher that in the dark ones.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1987

Kinetics and components of the flash photocurrent of isolated retinal rods of the larval salamander, Ambystoma tigrinum.

1. Membrane currents initiated by intense, 20 microseconds flashes (photocurrents) were recorded from isolated salamander rods by combined extracellular suction electrodes and intracellular tight-seal electrodes either in current or voltage clamp mode. The magnitudes (mean +/- 2 S.E.M.) of the maximal photoresponses recorded by the suction and by the intracellular electrode respectively were 40 +/- 5 pA (n = 18) and 35 +/- 7 mV (n = 8) for current clamp at zero current; 43 +/- 9 pA and 66 +/- 13 (n = 11) pA for voltage clamp at the zero-current holding potential, -24 +/- 3 mV. 2. Photocurrents initiated by flashes isomerizing 0.1% or more of the outer segment's rhodopsin achieved a saturated velocity and were 95% complete in less than 50 ms. The effect of incrementing flash intensity above 0.1% isomerization can be described as a translation of the photocurrent along the time axis towards the origin. Within the interval 0-50 ms the latter two-thirds of the velocity-saturated photocurrent is well described as a single-exponential decay. The decay was much faster in voltage clamp (2.8 +/- 1.2 ms, n = 11) than in current clamp mode (17 +/- 5 ms, n = 17). 3. The initial third of the velocity-saturated photocurrent, occurring over the interval from the flash to the onset of exponential decay, followed about the same time course in current and voltage clamp. The time interval occupied by this initial 'latent' phase decreased with increasing flash intensity and attained an apparent minimum of about 7 ms in response to flashes isomerizing 10% or more of the rhodopsin at ca. 22 degrees C. 4. The hypothesis that the decay of outer segment light-sensitive membrane current is the same in current and voltage clamp was supported by an analysis of the difference between outer segment currents measured successively in the two recording modes. First, the tail of the difference current decayed exponentially with a time constant approximately equal to R x C, where R and C are independently estimated slope resistance and capacitance of the rod. Secondly, the integral of the difference current, when divided by outer segment capacitance, closely approximated the hyperpolarizing light response measured under current clamp. Thus, displacement current accounted for the difference between photocurrents measured in current and voltage clamp.(ABSTRACT TRUNCATED AT 400 WORDS)

Ambystoma↗

PubMedDate not supplied

[Cyclic GMP-induced structural transitions of photoreceptor membranes].

The influence of cyclic GMP on the structural state of photoreceptor membranes was studied. The spin probe technique was used to obtain the dependences of microviscosity of lipid domains of membranes on cGMP concentration. It was shown that microviscosity of 5-doxylsteraric acid environment in rod outer segment, disc and plasma mambranes changed at concentrations of 2.10(7); 5.10(-6) and 6.10(-6). Non-cyclic GMP was ineffective. The ultrasound treatment of membraneous samples resulted on disappearance of the structural changes.

Animals↗

PubMed1991

Light damage in the rat retina: the effect of dietary deprivation of N-3 fatty acids on acute structural alterations.

We investigated the effect of depleting membrane docosahexaenoic acid (DHA, 22:6n-3) content through dietary deprivation of n-3 fatty acids on the susceptibility of the photoreceptors and pigment epithelium cells to acute light-induced changes. Male Sprague-Dawley rats were raised throughout gestation, lactation and up to the age of 8 weeks on semi-purified diets containing either safflower oil (SFO, n-3 deficient diet) or soybean oil (SO) as the sole source of lipids. A third group was switched at weaning from safflower oil to soybean oil (SFO/SO). Rats were maintained on a 12 hr/12 hr light/dark cycle in which the light level at the front of the cages was 5-10 lx. Light damage was produced by exposing dark-adapted animals to diffuse white fluorescent light of 700-800 lx for 30 min followed by 90 min of darkness. In order to study recovery from light damage, additional groups of SFO and SO rats were returned to dim cyclic light for 27 hr following bright light exposure. DHA content in retinal phosphatidylethanolamine and phosphatidylcholine was 65-75% lower in rats fed SFO than in rats fed SO. The decrease was compensated for by an increase in 22:5n-6, the total content of polyunsaturated fatty acids (PUFA) being similar in both the SFO and SO groups. The SFO/SO rats had DHA levels similar to SO animals, but 22:5n-6 remained elevated resulting in a slightly higher level of total PUFA. Severe rod outer segment (ROS) membrane disruptions were seen following bright light exposure in rats on the SO and SFO/SO diets. The appearance of these disruptions did not change significantly during more than 24 hr in dim cyclic light. In contrast, there were virtually no acute ROS lesions in the SFO group. Furthermore, there was a strong light-elicited disk-shedding response in the SO rats but not in the other two groups. The pigment epithelium of the DHA deficient retinas showed a significantly greater accumulation of large lipid droplets in the dark-adapted state. Notably, whole retina rhodopsin levels were 15% higher in the SFO than in the SO group. These results indicate that depletion of retinal DHA reduces the susceptibility of the rod outer segments to acute light damage and at the same time may alter visual pigment photochemistry and other photoreceptor and pigment epithelium functions.

Animals↗

PubMed2004

A novel tetraspanin fusion protein, peripherin-2, requires a region upstream of the fusion domain for activity.

Peripherin-2 (also known as peripherin/rds), a photoreceptor specific tetraspanin protein, is required to maintain normal cell structure through its role in renewal processes requiring membrane fusion. It is the first tetraspanin fusogen and has been shown to directly mediate fusion between disk membranes and opposing membranes to maintain the highly ordered structure of rod outer segments. Localized to the C terminus of human, bovine, and murine peripherin-2 is an amphiphilic fusion peptide domain (residues 312-326) and a highly conserved region upstream of this domain that we hypothesize is essential for fusogenic function. Our previous studies indicated that substitution of a threonine for a proline at position 296 within this highly conserved region enhanced fusion activity. In this study we wanted to determine whether this proline is essential with the introduction of three additional substitutions of proline with alanine, leucine, and glutamic acid. Wild type, P296T, P296A, P296L, and P296E mutants of peripherin-2 were expressed as His6-tagged full-length proteins in Madin-Darby canine kidney (MDCK) cells. All of the proteins were localized to intracellular membranes and detected as 42-kDa monomers by Western blot analysis. The wild type, P296A, and P296L assembled into core tetramers; in contrast the P296T and P296E formed higher order oligomers. Fusogenic activity of full-length protein expressed in MDCK membranes and purified protein reconstituted in model membrane liposomes was determined using fluorescence quenching techniques. Fusion activity was decreased in the P296L, P296A, and P296E mutants both in endogenous MDCK membranes and in model liposomes. Collectively, these results suggest that the proline at position 296 is necessary for optimal function.

Amino Acid Sequence↗

PubMed1993

A comparison of phagocytosis by the retinal pigment epithelium in normal and delayed amelanotic chickens.

The retinal pigment epithelium (RPE) performs a critical role in maintaining the integrity and renewal of the retinal system by clearing the shed photoreceptor outer segment debris through the process of phagocytosis. If phagocytosis is impaired, accumulation of the debris occurs leading to the degeneration of the retina. Phagocytosis, measured by polyvinyl toluene bead uptake is a normal cell activity exhibited by the embryonic retinal pigment epithelium even before complete differentiation of the neuronal layers. It is significant that embryonic pigment epithelium from either normal or delayed amelanotic (DAM) strain is competent in the process of non-specific particle uptake. In normal chickens non-specific phagocytic activity is observable as early as embryonic day 9 and continues through the rest of the developmental period and throughout adult life. The retinal pigment epithelium in DAM chickens exhibits early phagocytic activity at embryonic day 9 and then through day 16, but by day 19 this function is lost. This loss of phagocytic capacity in RPE of the DAM strain precedes and may be instrumental in the gradual retinal degeneration exhibited by these animals. Specific phagocytosis of rod outer segments (ROS) was examined in 4-week-old post-hatch normal and DAM RPE. Phagocytosis of ROS was greatly reduced in DAM RPE implying that the defect in the DAM is expressed in non-specific and specific phagocytosis. This study indicates that the DAM retinal system can now serve as an alternative and comparative model for studying different aspects of retinal degeneration.

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↗