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In spite of its pivotal role in visual transduction, very little is known about guanylate cyclase of retinal photoreceptor cells. The enzyme has not yet been purified principally because of the difficulty in solubilizing it. We report here a simple method for solubilization of 67% of the cyclase activity from the retinal rod disk membranes (RDM). With Nonidet P-40 as detergent, the solubilization of cyclase is favored by a high concentration of KCl and exclusion of manganese. The solubilized and the residual insoluble enzymes are both highly unstable but could be partially stabilized by dithiothreitol. They were both insensitive to calcium, calmodulin, and atrial natriuretic factor. They also responded similarly to varying the manganese concentration in the assay. For the activity in both fractions, the Km for GTP was about 230 microM, Line-weaver-Burk plots showed that substrate binding was cooperative, and Hill plots suggested that there are two substrate binding sites. Cumulatively, these observations showed that while the entire activity could not be solubilized, the solubilized and the residual insoluble activities probably belonged to the same enzyme. Partial purification resolved the solubilized enzyme into two activities refered to as enzymes 1 and 2. Both had substrate saturation kinetics similar to the solubilized enzyme and were inhibited competitively by inorganic pyrophosphate, one of the products of the cyclase reaction. The Ki for PPi for enzyme 1 was 70-100 microM and 150-200 microM for enzyme 2. cGMP at concentrations up to 800 microM had no influence on the activity of either enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

The subsecond mobilization of intracellular Ca2+ by IP3 was measured with rapid mixing techniques to determine how cells achieve rapid rises in cytosolic [Ca2+] during receptor-triggered calcium spiking. In permeabilized rat basophilic leukemia cells at 11 degrees C, more than 80% of the 0.7 fmol of Ca2+/cell sequestered by the ATP-driven pump could be released by IP3. Half of the stored Ca2+ was released within 200 ms after addition of saturating (1 microM) IP3. The flux rate was half-maximal at 120 nM IP3. Ca2+ release from fully loaded stores was highly cooperative; the Hill coefficient over the 2-40 nM range was greater than 3. The delay time of channel opening was inversely proportional to [IP3], increasing from 150 ms at 100 nM IP3 to 1 s at 15 nM, indicating that the rate-limiting step in channel opening is IP3 binding. Multiple binding steps are required to account for the observed delay and nonexponential character of channel opening. A simple model is proposed in which the binding of four IP3 molecules to identical and independent sites leads to channel opening. The model agrees well with the data for KD = 18 nM, kon = 1.2 X 10(8) M-1 s-1, and koff = 2.2 s-1. The approximately 1-s exchange time of bound IP3 indicates that the channel gating sites are distinct from binding sites having approximately 100-s exchange times that were previously found with radiolabeled IP3. The approximately 1-1s response time of [Ca2+] to a rapid increase in IP3 level can account for observed rise times of calcium spikes.

Development of rod outer segment (ROS) in the posterior area of the retina in normal (GR) and mutant Campbell rats with inherited retinal dystrophy was studied using transmission electron microscopy and morphometry. In both strains of rats primitive cilia appear first after birth and their number progressively increases up to postnatal day 9. The first ROS membrane disks (MD) appear at postnatal day 5. Originally MD are randomly oriented but later they acquire a regular arrangement. At day 7 MD occupy about 14% area of posterior retina in transverse sections in Campbell rats versus 7% in normal animals. By day 9 MD occupy about 35% area in the same region of the retina in both lines of rats. The retina of 15-day-old rats possesses the definitive number of differentiated ROS. The data obtained show that during the period between birth and eyelid opening ROS morphogenesis in Campbell rats is not slow or disturbed as compared with that in normal rats.

Rod outer segment (ROS) phagocytosis by explanted bovine retinal pigment epithelium (RPE) was evaluated by a procedure using an indirect double radioimmunoassay which distinguished between ROS attached to the RPE cell surface and those which had been ingested. This approach has been used to investigate the effect of a variety of glycoconjugates on the phagocytic process. Inclusion of the glycosaminoglycans (GAGs) chondroitin sulphate type-A (CS-A) and type-C (CS-C), hyaluronic acid (HA) or dermatan sulphate (DS) in the incubation medium significantly inhibited the ingestion phase of ROS phagocytosis, whereas the binding phase was inhibited to a lesser extent. The interphotoreceptor matrix (IPM), containing these GAGs as part of proteoglycans, also had an inhibitory effect on phagocytosis. The free monosaccharides mannose, fucose and galactose all stimulated the ingestion of ROS by RPE cells. These findings support the suggestion that glycoconjugates may have a physiological role in the photoreceptor renewal process.

The retina of the common mole, Scalopus aquaticus, has been studied with the transmission electron microscope. Structures examined include: pigment epithelium, outer and inner segments of the sensory cells, and synaptic ribbons of the outer plexiform layer. Rods and cones described in species by previous light microscopic studies are not seen with electron microscopic techniques. Instead, the mole retina contains peculiar outer segments consisting of whorls of membranes. These whorls have some similarities to dystrophic retinas of several vertebrates. The possibility of their being caused by extraordinary light exposure is discussed, also. The appearance of the sensory cells suggests that they are functional receptors.

When pure, synthetic lipid hydroperoxides (LHP) were injected into the vitreous body of albino rabbits, electrical activity was decreased in all components of the electroretinogram (ERG) in a progressive and time-related manner. In the early phase morphological changes occurred at the interface between the photoreceptor outer segments and the retinal pigment epithelium (RPE) in response to LHP. These findings commenced within a few hours after injection and continued during the following 2-3 weeks, when the ERG was completely extinguished. Two days after injection, the rod outer segments (ROS) were swollen and damage of the RPE apical villi was observed. This initial event was followed by additional changes in the RPE which embraced swelling, accumulation of residual bodies, complete loss of ROS and enlargement and disruption of Bruch's membrane. The precursors of residual bodies appeared to result from focal, peroxidative damage to ROS discs which apparently rendered these materials undegradable by the RPE. As ROS degeneration continued, the RPE showed hypertrophy and modification. These studies provided evidence for a sequential destruction of the neural retina and RPE during oxidative damage involving lipid peroxidation. The mechanism appeared to differ from that produced by other toxic compounds or those which resulted from vitamin E or A deficiency. This new model system is thought to be useful in 1) explaining differences in susceptibility of inner ROS disks versus other membranes, 2) determining how the RPE metabolizes abnormal ROS, 3) studying RPE reactivity following trauma and/or retinal detachment, and 4) determining factors which produce degeneration of Bruch's membrane.

The retinas of six pigmented rabbits (12 eyes) were irradiated with a cw-Nd:YAG laser using two different pulse forms, in conjunction with a constant pulse duration of 200 msec and a retinal focus diameter of 200 microns. The first pulse form (a) was characterized by an initial transient, lasting 15-20 msec, with a peak power twice that of the ensuing plateau phase of 180-185 msec. The second pulse form (b) was almost rectangular in shape. Although the average energies (and powers) for each pulse form were identical, almost 20% of this entity was delivered within the first 20 msec for the first pulse form. Impact lesions were examined at the biomicroscopic, light, and electron microscopic levels, and the characteristic morphologic features described for the cw-mode in general. Comparison of the effects produced using each pulse form revealed a number of differences. At low pulse energies (less than 150 mJ) the rectangular pulse form produced a photocoagulation reaction within the outer sensory retina, but when using the pulse form with the initial transient, this effect was superimposed by a degree of mechanical disruption. Ruptures produced within the elastic component of Bruch's membrane first appeared at lower pulse energies (ie, at 75 mJ, as opposed to 150 mJ for the rectangular pulse form) and were more frequent, and choroidal macrohemorrhaging (observed at pulse energies greater than or equal to 200 mJ) occurred only, when this latter pulse form was employed. Each of these phenomena may be attributed to the higher power and energy densities attained during the initial transient, which has disruptive properties. In order to avoid the risk of severe bleeding, these irradiation parameters should be maintained below approximately 6.4 X 10(3) W/cm2 and approximately 0.13 X 10(3) J/cm2.

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

A technique by which the retina can be isolated from the turtle eye is described. Scanning electron microscopy revealed morphological variability between preparations and also between regions of the same one. Large areas were often totally free of any pigment epithelial cells, yet contained a high proportion of photoreceptors with complete outer segments. However, adjacent regions may contain photoreceptors without outer segments or with fragmented ones. The physiological properties of the horizontal cells also demonstrated large variability between different preparations. In all cases, lowering calcium concentration from 2 mM to 0.1-0.5 mM depolarized the horizontal cells and augmented the amplitude of the maximum photoresponses. However, these effects were accompanied by changes in the photoresponse kinetics and by a reduction in the horizontal cell sensitivity to light. Moreover, prolonged exposure to low calcium induced permanent damage to the retina as was indicated by the reduction in the response amplitude after superfusion with 2 mM calcium solution had been resumed. The toxic effects of low calcium were most apparent when superfusion with 0.1-1.0 microM calcium concentration was performed. These solutions induced complex time-dependent effects on the resting potential of horizontal cells and on the amplitude and kinetics of the photoresponses. We conclude from these observations that the normal concentration of extracellular calcium in the turtle retina is in the 2 mM range.

This report describes the ultrastructural localization of S-antigen in pig and human retinas by means of a highly specific rat IgG monoclonal antibody, S2.4.C5, followed by a secondary antibody adsorbed to colloidal gold. This monoclonal antibody gave definitive staining with negligible background. The protein was detected in both the rod outer and inner segments. Connecting cilia and the rod outer segment disc membranes were labelled. The outer segment plasma membrane was not obviously labelled. Cones were labelled at background level. S-Antigen was not detected in any other cells of the neural retina. The fate of S-antigen was also followed to the pigment epithelial phagosomes where intracytoplasmic ROS debris was stained with the antibody. No label, however, was detected in the choroid, suggesting that trans-cellular transport of the S-antigen did not occur.

Monoclonal antibodies were used with radioimmune assays and immunocytochemical techniques to identify and localize membrane proteins in bovine rod outer segment (ROS) disc membranes. When ROS membrane proteins were separated by SDS-polyacrylamide gel electrophoresis in the presence of the sulfhydryl reducing agent, 2-mercaptoethanol, two monoclonal antibodies designated as 3B6 and 2B6 were found to bind to a polypeptide having an apparent molecular weight (Mr) of 33,000 daltons. In the absence of 2-mercaptoethanol, these monoclonal antibodies bound to a doublet having Mr of 67,000 and 69,000. Immunogold-dextran labeling of ROS sections embedded in Lowicryl resin indicated that this protein is localized around the periphery of the ROS organelle where the discs come in close contact to the ROS plasma membrane. Immunogold labeling of morphologically intact isolated discs prepared by mild trypsinization of ROS fragments confirmed that this disc membrane protein is localized along the rim region of discs. On the basis of these localization studies, the authors have named this protein peripherin. Immunogold-dextran markers were also used with previously characterized antirhodopsin monoclonal antibodies to visualize the distribution of rhodopsin on isolated discs. Dense labeling was observed along the lamellar region of the discs, but little if any labeling was observed on the extreme edges of the discs. These results are consistent with the view that the lamellar region of discs containing rhodopsin is a distinct membrane domain from the rim region which contains peripherin, a high Mr rim protein and possibly other proteins involved in disc-disc and disc-plasma membrane interactions.

The external photoreceptor segments in the retina of vertebrates have been studied already more than 100 years; the intensity of these studies increased markedly during the recent 20 years due to the introduction of autoradiography and electron microscopy. These methods allowed to study the ultrastructure of external segments of the rods and cones in many vertebrate species, its development in ontogenesis, the renewal of external segments and the dependence of renewal on the diurnal rhythm of illumination. All these data have never been summed up and discussed. This review provides a general picture of studies of different aspects of the ultrastructure of external segments; the models of formation of the external segments' discs proposed by several authors are considered; the necessity of continuation and widening of comparative studies of the ultrastructure of external photoreceptor segments in different vertebrates is founded. The most important conclusions concern the differences between the rods and the cones during renewal of the membranes discs of external segments.

The visual pigments, and the structure of the visual cells, were investigated by spectrophotometry and by light and electron microscopy in fetal and adult sheep. The rhodopsin system in adult sheep closely resembles that of cattle. The absorbance maxima of rhodopsin, lumirhodopsin, and metarhodopsin I are at 498, 490, and 480 nm, respectively. The estimated molar absorbance coefficient at the wavelength of maximum absorption of rhodopsin is 40,000M-1 . cm-1. Rhodopsin was detected from a fetal age of 85 days (term at 145 days). Partial bleaching of extracts from fetal eyes (95, 105, and 115 days) did not demonstrate cone pigments, although cones were present in fair numbers at a fetal age of about 105 days. The time course of rhodopsin formation between 85 and 140 days resembles a growth curve. The amount of rhodopsin shortly before birth (140 days) is about 0.6 times that in the adult. The number and dimensions of rod outer segments as well as packing of the discs were studied structurally and related to the rhodopsin content. A fairly good correlation was found at the earliest stages (95 and 105 days gestation age), when rhodopsin concentration was very low and rod outer segments were few and small, as well as at the latest stage (140 days). At 115 days the rhodopsin content observed by spectrophotometry was less than that indicated by the outer segment volume, probably mainly due to the outer segment discs and possibly to the rhodopsin molecules being less tightly packed than at 140 days.

Proteolysis of rhodopsin in disc membranes of right-side out orientation by thermolysin, papain and St. aureus V8 protease allowed to identify two highly exposed regions of polypeptide chain located on the cytoplasmic membrane surface: carboxyl terminal sequence 321-348 and the fragment 236-241. Incubation with chymotrypsin reveals the third site on the cytoplasmic surface, 146-147, accessible to proteolytic enzymes. Frozen-thawed membranes comprise a mixture of vesicles with normal and inverted orientation. Both thermolytic and chymotryptic digests of rhodopsin in these membranes contain the polypeptide which represents the amino terminal sequence lacking the first 30 amino acid residues. Thus at least 30 amino acids from the N-terminus must protrude into the intradiscal space. One additional site was located on the intradiscal surface: papain digests rhodopsin in the inverted membranes at the position 186-187. Localization of the proteolytic cleavage sites allowed to propose a model for rhodopsin topography in disc membrane: the polypeptide chain traverses the bilayer thickness seven times; each of seven transmembrane segments containing approximately 40 amino acid residues includes a sequence of approximately 30 hydrophobic amino acids; which are probably in close contact with hydrocarbon matrix of the membrane. Hydrophobic sequences are terminated with fragments containing clusters of hydrophilic amino acids, possibly interacting with lipid polar head groups and orienting each segment in the bilayer.

Ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K- NPPase ) activity of the Na-K-ATPase complex and the Ca-ATPase activity were studied ultracytochemically in the inner and outer segments of guinea pig photoreceptor cells by means of newly developed methods. The K- NPPase activity was demonstrated on the plasmalemma of the inner segments, whereas the Ca-ATPase activity was restricted to the matrix side of the inner membrane of the mitochondria which were accumulated in the inner segments. In contrast to the enzyme activities in the inner segments no such activity could be detected on the plasma and disk membranes of the outer segments. The functional meaning of the enzyme activities is discussed.

The retina of Xenopus laevis contains two populations of rod photoreceptors that differ in their utilization of L-fucose. Following intraperitoneal injections with 3H-fucose, all rod and cone photoreceptors incorporate the label. One day after labeling, much of the radioactivity is associated with the photoreceptor outer segments. In rods, a band of radioactivity is initially located at the base of the outer segment. As the time interval between injection and recovery is extended, the band of radioactivity is progressively displaced toward the outer segment tip. When the autoradiography exposure times are reduced so that cone and most rod outer segments no longer appear labeled, a minor population of rod photoreceptors can be distinguished which remains heavily labeled. The outer segment of the principal rod is 29.8 +/- 0.6 micrometers long and 6.4 +/- 0.6 micrometers in diameter, whereas the outer segment of the minor rod is 19.7 +/- 3.4 micrometers long and 4.5 +/- 0.6 micrometers in diameter, the latter accounting for approximately 2-3% of the total rod photoreceptor population. The rate of 3H-band displacement is 2-fold greater in the minor rod outer segment than the renewal rate of the outer segment in the principal rod. The similarity in relative cell density and size of the minor rod suggests that this photoreceptor corresponds to the blue-sensitive rod of Xenopus recently described by Witkovsky et al. (1981a, b), Vision Res. 21, 863-867, 875-883).

Foetal retina was removed from donor rats at 15 days of gestation and transplanted to the occipital cortex of neonatal host rats. The purpose of this procedure was to examine the development of retinal neurons and photoreceptors, and document synaptic patterns during maturation of the transplanted retina in an environment lacking a normal target for optic axons. Host animals were sacrificed at 5, 10, 15, 20 and 30 days and samples of cortex containing the transplant were subjected to a light and electron microscopic analysis. During early stages of development, (5 days) the retina assumes a radial orientation with the scleral (outer) surface located centrally and the vitreal (inner) surface occupying the periphery. Numerous mitotic figures are found at the centre of the transplant and columns of primitive neuroblasts appear to radiate out from this zone. By 10 to 15 days after transplantation the retinal tissue contains numerous small rosettes each of which displays a histotypic organization with recognizable layers of sensory cells and their centrally-projecting processes, an outer limiting membrane, made up of a network of zonulae adherentes, and a rudimentary outer and inner plexiform layer which delineate the cells of the inner nuclear layer. Ultrastructural analysis of such rosettes confirmed the presence of typical bipolar, amacrine, horizontal and ganglion cells, but revealed that while the plexiform layers were occupied by numerous processes from these neurons, few if any, of these exhibited synaptic vesicles. By 20 to 30 days following transplantation sensory cells have completely differentiated, giving rise to prominent inner and outer segments which display typical cilia, centrioles and basal bodies, together with numerous stacked lamellae of photoreceptors which were contorted, presumably due to growth in an abnormal site. It should be further emphasized that these structures developed in the absence of pigment cells. Synaptic development ensues during this period to form characteristic dyads within the outer and inner plexiform layers. Additionally, clusters of amacrine to amacrine contacts occurred in the inner plexiform layer and were found to be increased relative to other types of junctions. In general, synaptogenesis takes place in the outer and inner plexiform layers and all categories of retinal synapses are established, but the process was found to be significantly delayed in comparison to normal retina at the same stage of development. Quantitative analysis revealed a reduced number of presumptive ganglion cells in proportion to the other categories of neurons. Optic fibres remained small and failed to myelinate. It is suggested that lack of an appropriate target for optic axons induced this alteration and may be indirectly related to the delay in the onset of synaptic development.

Moxalactam disodium is a new third-generation semisynthetic, broad-spectrum, cephalosporin-like antibiotic for parenteral administration. Topical, subconjunctival, and intravenous administration provide poor concentration in the vitreous. To determine its toxicity in intravitreal administration, we injected comparative doses directly into the vitreous cavity of 21 rabbits. With doses of 1.25 mg or less there was no toxic damage to the retina. With a dose of 2.5 mg, early degeneration of photoreceptors was seen after three months. With higher doses (5 and 10 mg) there were major histopathologic and electroretinographic changes. These results suggest the feasibility of employing moxalactam in the treatment of acute, severe, fulminant bacterial endophthalmitis.

It has been postulated that ascorbic acid may help to protect the retina from oxidative insult by light. To confirm this hypothesis, the authors compared light-damaged retinas of rats with or without ascorbate supplement by morphologic and morphometric studies at different time periods after light exposure. No dramatic morphologic differences were observed in the photoreceptor-retinal pigment epithelium complex between the two groups six hr after light exposure to 200 to 250-foot candles of visible light. Six to 13 days after 24 hr of exposure, the retina of rats that received ascorbate supplement showed significantly less severe damage than the retina of unsupplemented rats. The superior and temporal quadrants of the retina appeared to be most susceptible to the light damage when comparing rats with or without ascorbate supplement. These findings suggested that ascorbate ameliorates the photic injury in rat retina.

Immunoreactive opsin was detectable in the apical portion of normally developing photoreceptor cells on postnatal day 3 by the indirect enzyme-labeled antibody method. Immunoreactivity increased and had extended from the central retina to the periphery by the advanced stages of development. In the rd mutant retinas, accumulated opsin was present in the apical portion and in the outer nuclear layer on postnatal day 8. Immunoreactive opsin mainly was present in the outer nuclear layer by day 14, even being detectable on day 28. No immunoreactivity was present in the remaining cones. Electron microscopic immunocytochemistry confirmed the association of immunoreactive opsin with the persistent rod cell plasma membrane. Molecular weight of immunoreactive opsin in 14-day-old rd mutant mouse retina, as estimated by gel filtration chromatography, was large and did not seem to be degraded. These findings indicate that accumulated rhodopsin continues to function in the plasma membrane because an electroretinogram could be made after day 14 for the rd mutant mouse retina.

The effects of moderate-intensity light (150 to 200 ft-cd) on retinal structure were compared between retinol-adequate and retinol-deficient rats after 1 to 6 days of light exposure during the 12 hr light phase of the cycle. Both damage to the outer segments and loss of photoreceptor cells were accelerated in retinol-adequate rats. Outer segments in retinas of retinol-adequate rats showed an abnormal staining pattern and disruption of disc structure in the distal portion about 2 days before those of retinol-deficient rats. After 4 days of exposure 24% of the photoreceptor cells had degenerated in the retinol-adequate retinas, but only 6% in the retinol-deficient retinas. By 6 days 65% and 41% of the photoreceptors had degenerated in the retinol-adequate and retinol-deficient retinas, respectively. Thus light exposure induced more rapid degeneration of photoreceptor cells in rats receiving adequate retinol than in those deficient in this vitamin.

Frog retinas were examined following cryoultramicrotomy. The extent of glutaraldehyde cross-linking ranged from maximally 2% for 2 h to no cross-linking. When the tissue was cryoprotected, the effects of hypotonic, isotonic and hypertonic media on outer segment banding periodicities were studied. The specimen was cryosectioned at 193 K to 113 K with sections collected on a dry knife (213 K to 123 K). The sections were freeze-dried and only rehydrated with phosphotungstic acid section stain when transmission electron microscopy was used to establish the extent of gross retinal morphology for each fixation protocol employed. As the extreme of no cross-linking and no cryoprotection was approached and reached, conventional sections which displayed extensive retinal morphology were not obtained. Instead, fragments of retinal tissue resulted which showed periodicities of alternating light and dark bands. These periodicities are interpreted as photoreceptor outer segment discs with contrast formation depending upon an interplay between three factors: mass density differences between disc and interdisc space, section cutting angle, and section thickness.

Mg2+-ATP produces a large decrease in near-IR light scattering when added to suspensions of rod outer segments (ROS) when the plasma membranes have been disrupted by a gentle dialysis procedure. When this process is studied by light microscopy with video-enhanced image contrast, the Mg2+-ATP-dependent signal is seen to be associated with the formation of filaments which extend only from those ROS lacking plasma membranes. Both the IR light scattering signal and filament growth are inhibited by vanadate and DCCD but not by colchicine, colcemid or cytochalasins.

Opsin-immunoreactive sites in the eye and optic nerve of the hagfish, Myxine glutinosa, were studied by use of light-microscopic pre- and postembedding peroxidase-antiperoxidase or avidin-biotin-peroxidase techniques, and the immuno-electron-microscopic protein A-gold method. At the light-microscopic level, a strong opsin immunoreaction was obtained on the outer segments of the photoreceptor cells with sheep and rat antibodies against bovine (rhod)opsin. These outer segments were located in the marginal photoreceptor space and in follicles of the retina, as well as in the tubular lumen of the optic nerve. Ultrastructurally, two classes of outer segments can be distinguished; most of them exhibited a strong antiopsin reaction, while certain elements lacked immunoreactivity with the antisera employed. The protein A-gold particles marked opsin-immunoreactive sites on the photoreceptor membranes. The presence of opsin-immunoreactive material in the retina and optic nerve of the hagfish strengthens the view that this primitive eye lacking a cornea, lens and vitreous body is engaged in light perception. The morphological similarity between the eye and pineal tissue is discussed in connection with the absence of a pineal organ in this species.

In the heterozygous mutant (rds/+) mice, receptor outer segments (ROS) are irregular in form and are shed as abnormally large phagosomes. In the albino rds/+ mice, peak frequency of pigment epithelial (RPE) phagosomes is higher than normal and is recorded near the end of the light period, instead of at the time of light onset as in the normal (+/+) albino mice. In pigmented mice of both genotypes, the maximum numbers of phagosomes in the RPE remain lower than in the albinos. In pigmented +/+ mice the number of phagosomes is already high at the time of light onset. The number rises to peak after one hour and then declines slowly. The lowest frequency is reached after the end of the light period. In pigmented rds/+ mice, the number of phagosomes in the RPE is lowest at the time of light onset. The number rises rapidly to peak level within two hours, then declines and remains low until light onset. If the dark period is prolonged, phagosome frequency in the rds/+ RPE remains lower than in +/+ RPE. If the light period is prolonged, phagosome frequency in the rds/+ RPE remains at a higher level than in the +/+ RPE. This differential response to altered light regimen in the rds/+ and +/+ mice is less pronounced in the pigmented than in the albino individuals. The phagosomes in the rds/+ RPE are larger than in the +/+ RPE in all light regimens. These results show that ocular pigmentation may modify the circadian pattern of ROS disc shedding in the rds/+ retina.

The purity criteria of bovine rod outer segments (ROS) purified by different procedures were evaluated. Bovine ROS were purified by flotation and/or sedimentation in a continuous concentration gradient of sucrose. The purity of the different fractions was then evaluated according to four purity criteria: (i) the A280/delta A500 ratio, (ii) the moles of phospholipid per mole of rhodopsin, (iii) the fatty acid composition, and (iv) the interfacial properties of ROS membranes. All the purity criteria, except the A280/delta A500 ratio, were found to be adequate. From our results, the A280/delta A500 ratio cannot be used alone to characterize ROS purity. Furthermore, the phospholipid-to-rhodopsin ratio appears as the best purity criterion because of its reliability, its higher sensitivity, and its ease of achievement. It is noteworthy that mechanical treatment of the retinas dramatically affects the purification of ROS.

The sequential electrophysiologic and pathologic changes in the retina in ceroid-lipofuscinosis were recorded in a time-course study in the ovine model. Over a relatively short period in the course of the disease, a severe reduction in both rod and cone b-wave amplitudes developed with rod b-wave changes preceeding those of cones. These changes paralleled a similar loss of rod and cone photoreceptor cells. In affected retinas, outer segments appeared shorter than normal. By 84 weeks of age, the outer nuclear layer was reduced to the width of a single nucleus. In addition to these changes, electronmicroscopy showed the formation of abnormal dystrophic rod and cone outer segments in photoreceptor cells. Most cells in the retina showed the accumulation of a fluorescent lipopigment, this being most prominent in ganglion cells. Ultrastructural studies showed them to be made of electron dense granular material and a variety of membranous and tubular arrays.

We performed electron microscopy of replicas from freeze-fractured retinas exposed during or after fixation to the cholesterol-binding antibiotic, filipin. We observed characteristic filipin-induced perturbations throughout the disk and plasma membranes of retinal rod outer segments of various species. It is evident that a prolonged exposure to filipin in fixative enhances rather than reduces presumptive cholesterol detection in the vertebrate photoreceptor cell. In agreement with the pattern seen in our previous study (Andrews, L.D., and A. I. Cohen, 1979, J. Cell Biol., 81:215-228), filipin-binding in membranes exhibiting particle-free patches seemed largely confined to these patches. Favorably fractured photoreceptors exhibited marked filipin-binding in apical inner segment plasma membrane topologically confluent with and proximate to the outer segment plasma membrane, which was comparatively free of filipin binding. A possible boundary between these differing membrane domains was suggested in a number of replicas exhibiting lower filipin binding to the apical plasma membrane of the inner segment in the area surrounding the cilium. This area contains a structure (Andrews, L. D., 1982, Freeze-fracture studies of vertebrate photoreceptors, In Structure of the Eye, J. G. Hollyfield and E. Acosta Vidrio, editors, Elsevier/North-Holland, New York, 11-23) that resembles the active zones of the nerve terminals for the frog neuromuscular junction. These observations lead us to hypothesize that these structures may function to direct vesicle fusion to occur near them, in a domain of membrane more closely resembling outer than inner segment plasma membrane. The above evidence supports the views that (a) all disk membranes contain cholesterol, but the particle-free patches present in some disks trap cholesterol from contiguous particulate membrane regions; (b) contiguous inner and outer segment membranes may greatly differ in cholesterol content; and (c) the suggested higher cholesterol in the inner segment than in the outer segment plasma membrane may help direct newly inserted photopigment molecules to the outer segment.

Although there have been several reports pertaining to the existence of the cGMP-gated channel in the disk membrane of rod photoreceptors, its density there relative to that of the photoreceptor plasma membrane is unknown. Using immunoblotting, immunohistochemical, and reconstitution techniques on purified disk and plasma membrane preparations, we found that the density of channels in the plasma membrane was at least 50-fold higher than that of the disk membrane. Purification of membrane fractions without prior digestion of cytoskeletal components by mild trypsinization was found to increase the amount of channel protein present in disk membrane preparations. We propose that the presence of the channel protein in rod disk membrane preparations is an artifact arising from fusion of plasma membrane components during permeabilization of the photoreceptor cell.

The effect of occlusion of the vortex vein to the retinal pigment epithelial cells (RPE) and the outer segments of the rod visual cells (OS) of albino rabbits was observed by scanning electron microscopy. Three vortex veins of the left eye and two vortex veins of the right eye in each rabbit were ligated with 6-0 silk suture. The morphological changes were observed at 24 hours, 1, 2, and 4 weeks after the ligation. The changes of RPE showed no significant difference between ligation of 2 and 3 vortex veins. The RPE showed degenerative changes 1 week after the ligation and the degenerative changes became severe after 2 weeks. The OS, showed no change after 2 vortex veins occlusion, while after occlusion of 3 vortex veins OS revealed degenerative changes after 1 week and the degenerative changes reached a maximum after 2 weeks. The changes of both RPE and OS tended to return to normal at 4 weeks. The changes of both RPE and OS may be caused by reduction of the blood flow in the choroidal circulation.

We studied both eyes of a 66-year-old man with retinal degeneration and oat cell carcinoma of the bronchus. Retinal degeneration was most marked peripheral to the parafovea where photoreceptor cells and their outer segments were absent. Within the parafovea, photoreceptor cells remained but rod outer segments were absent and cone outer segments were fragmented and disorganized. The retinal pigment epithelium contained many immature melanin granules within melanolysosomes, suggesting abnormal melanin synthesis and resorption. We suggest that a pharmacologically active substance resembling a hormone produced by the tumor increased melanin synthesis in the pigment epithelium and that the increased melanin content in these cells compromised their ability to phagocytose and maintain normal turnover of photoreceptor outer segments. We believe these changes led to photoreceptor outer segment loss and subsequent degeneration of the photoreceptor cells.

The temporal pattern of cone outer-segment disc shedding was examined in the retina of the California ground squirrel, Spermophilus beecheyi, under two different lighting conditions. Squirrels were entrained to either 10-180 lx (room lighting) or 1,400 lx. Cone shedding during the dark period was biphasic in both conditions, with increases occurring at 2-3 hr and 5-6 hr after light offset. Entrainment to 1400 lx resulted in an increase in shedding at 2 hr after light offset and a slight advance in the timing of both peaks. Dense granules were often found in photoreceptors, retinal neurons, Müller cells, microglia and vascular cells. These granules, which were found primarily during the dark period and early light period, were lipofuscin-like in their lipophilia, size and autofluorescence. Many of the granules were probably autophagic in origin, but some within Müller cells may have originated via endocytosis of extracellular material which was exocytosed by photoreceptors.

The European eel (Anguilla anguilla) has a complex life cycle during which it changes habitat and to some extent morphology. During this time the eye undergoes modifications in both size and photopigments. This report forms the first of a comparative morphological study of the retina of the eel during the major stages of its life cycle. The retinal pigment epithelium of the glass eel stage consists of a single layer of cuboidal cells joined laterally by cell junctions. Both the basal (scleral) and lateral borders of these cells are relatively smooth while apically (vitreally) numerous processes enclose photoreceptor outer segments. Within the epithelial cells are abundant mitochondria, melanin granules, lipid droplets, myeloid bodies and a peculiar "multitubular body". Phagosomes are present but not numerous. Bruch's membrane is relatively thin and homogeneous. The choriocapillaris although present is not widespread and displays typically fenestrated endothelium.

To study precursor-product relationships between cytoplasmic membranes of the inner segment of photoreceptors and the continually renewed outer disc membrane, we have compared the density and size distribution of intramembrane particles (IMP) in various membrane compartments of freeze-fractured photoreceptor inner and outer segments. Both rod and cone outer segments of Xenopus laevis are characterized by a relatively uniform distribution of approximately 4,400-4,700 IMP/micron2 in P-face (PF) leaflets of disc membranes. A similar distribution of IMP is found in the outer segment plasma membrane, the ciliary plasma membrane, and in the plasma membrane of the inner segment in the immediate periciliary region. In each case the size distribution of IMP can be characterized as unimodal with a mean diameter of approximately 10 nm. PF leaflets of endoplasmic reticulum, Golgi complex, and vesicles near the cilium have IMP with a size distribution like that in the cilium and outer segment, but with an average density of approximately 2,000/micron2. In contrast, IMP are smaller in average size (approximately 7.5 nm) in PF leaflets of inner segment plasma membrane, exclusive of the periciliary rgion. The similarity of size distribution of IMP in inner segment cytoplasmic membranes and those within the plasmalemma of the cilium and outer segment suggest a precursor-product relationship between the two systems. The structure of the vesicle-rich periciliary region and the segregation of IMP with different size distributions in this region suggest that components destined for incorporation into the outer segment exist as preformed membrane packages (vesicles) which fuse with the inner segment plasma membrane in the periciliary region. Subsequently, membrane components may be transferred to forming discs of the outer segment via the ciliary plasma membrane.

Nineteen domestic cats were entrained to a 12-hr light/12-hr dark lighting cycle and killed at different times of the cycle. The numbers of rod and cone phagosomes in the retinal pigment epithelium (RPE) were quantified by light microscopy. The number of both rod and cone phagosomes shows a large increase within 2 hrs after light onset. Thereafter, the phagosome count remains low until the latter part of the dark period when the number of rod and cone phagosomes increases slightly. In a few cases, substantial variability in phagosome numbers was found between animals killed at the same times in the lighting cycle. Small (0.4-0.8 microns), dense, membrane-bound granules, that have the ultrastructural features of lysosomes, occur in large numbers in the tapetal RPE cells. The granules may contain only a homogeneous matrix or matrix plus membrane fragments or melanin. Variations in the number of such granules in different animals shows no clear diurnal pattern. Phagosome number, size, and length measurements of outer segments are used to provide estimates of cone outer segment turnover time in the cat.

31P NMR studies of rod outer segment (ROS) and sarcoplasmic reticulum (SR) membranes have been performed under conditions where broad and narrow spectral components can be clearly resolved. Control studies of an anhydrous, solid powder of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), as well as aqueous binary mixtures of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), demonstrate clearly that broad spectral components can be detected. For the codispersions of DSPC and DOPC in the mixed-phase region at 22 degrees C, the 31P NMR spectra consist of a superposition of a broad component and a narrow, axially symmetric component, due to coexisting solid and liquid-crystalline domains, which are in slow exchange on the 31P NMR time scale. The 31P NMR spectra of the native ROS and SR membranes, however, consist of only a narrow component, to within experimental error, indicating that most or all of the phospholipids are in the liquid-crystalline (L alpha) phase at 22 degrees C. The above conclusions are in agreement with many, but not all, previous studies [see, e.g., Yeagle, P.L. (1982) Biophys. J. 37, 227-239]. It is estimated that at most 10% of the phospholipids in the ROS and SR membranes could give rise to broad 31P NMR spectral components, similar to those seen for anhydrous or solid-phase lipids, corresponding to approximately 7 phospholipids/rhodopsin molecule and approximately 11 phospholipids/Ca2+-ATPase molecule, respectively.

The rod outer segment (ROS) is attached to the visual cell body by a connecting cilium. Axonemal components of this cilium extend into the ROS, an organelle which undergoes continuous renewal throughout life. New membranous disks are added at the ROS base, while older ones are shed from the tip. The formation of new disks is believed to result from plasma membrane evaginations at the distal end of the connecting cilium, but the mechanism responsible for disk morphogenesis is not yet understood. Within the ciliary axoneme, at the base of the ROS, an actin-rich domain has been localized with immunoelectron microscopy, and filamentous actin has been detected with fluorescent phallotoxin. However, actin filaments have not previously been observed in electron micrographs of this region. We now report that a meshwork of decorated actin filaments was observed within the center of the ciliary axoneme, at the base of the ROS, after visual cells were permeabilized with saponin and incubated with myosin subfragment-1 (S-1). Furthermore, individual filaments were seen to extend from the center of the axoneme into the base of the ROS disk stack by passing between pairs of ciliary microtubule doublets. Arrowheads on these filaments uniformly pointed toward the cilium, while the barbed (or fast-growing) ends were oriented in the direction of disk expansion and were often associated with the ROS plasma membrane. In control retinas, undecorated filaments were observed. Thus, S-1 binding did not induce filament formation. These results suggest that an actin filament network may provide cytoskeletal support and guidance for the growing ROS disks.