Diurnal expression of transducin mRNA and translocation of transducin in rods of rat retina

The messenger RNA (mRNA) that encodes alpha subunit of the guanosine triphosphate-binding protein transducin (T alpha) and T alpha immunoreactivity were localized and measured in the rat retina during the light-dark cycle with in situ hybridization and immunohistochemistry. Both T alpha mRNA and T alpha immunoreactivity were observed only in photoreceptors. Within the photoreceptor T alpha mRNA was present primarily in the inner segments and to a lesser extent in the outer nuclear layer at all times during the day and night. However, the distribution of T alpha immunoreactivity varied profoundly with the light-dark cycle; during the day, T alpha immunoreactivity was highest in the inner segments, and at night the outer segments were more immunoreactive. The amounts of T alpha mRNA and T alpha immunoreactivity also depended on the light-dark cycle. Levels of T alpha mRNA were high immediately before and after lights on; levels were low for the rest of the light-dark cycle. During the day, T alpha immunoreactivity increased in the inner segments following the increase in T alpha mRNA. After the lights were turned off, T alpha immunoreactivity decreased in the inner segments and increased in the outer segments. Thus, it appears that T alpha is synthesized in the inner segments after a morning increase in T alpha mRNA. Newly synthesized T alpha remains in the inner segments until it is transported to the outer segments at night, where it may be involved in the increase in the sensitivity of photoreceptor rods at night.     

Intracellular topography of rhodopsin bleaching

In a vertebrate eye, the photoreceptor cells are aligned so that most of the light passes through them lengthwise. At the light-transducing outer segment region of the photoreceptor, photons are absorbed in a time-varying, spatially dependent fashion. Because the transduction event is spatially localized around the site of photon absorption, the spatiotemporal patterns of light absorption in outer segments are an important receiver input characteristic. This aspect of receptor biophysics has now been measured; the results were consistent with a theoretical model proposed for bleaching of a pigment in an unstirred layer.     

Digitonin effects on photoreceptor adenylate cyclase

Adenylate cyclase is described in a number of photoreceptor membranes. Vertebrate rod outer segments contain light-regulated cyclase, and light regulation is abolished by digitonin. Disruption of microvilli in cone and rhabdomphotoreceptors is also associated with loss of light regulation and retention of full enzymic activity. The data suggest that inhibitory constraint provides regulation in cyclase systems and that disruption of membrane structure uncouples catalytic and regulatory elements.     

Identification of specific transducin alpha subunits in retinal rod and cone photoreceptors

Transducin is a guanyl nucleotide-binding protein that couples rhodopsin photolysis to hydrolysis of guanosine 3',5'-monophosphate in rod photoreceptor cells of vertebrate retinas. Several complementary DNA clones encoding transducin subunits have recently been characterized. One clone, isolated from a bovine retina complementary DNA library, encodes a previously unidentified polypeptide with an amino acid sequence 78% identical to the sequence of the alpha subunit of bovine rod outer segment transducin. Antibodies to a synthetic peptide with amino acid sequence derived specifically from this novel polypeptide recognize a 41-kilodalton polypeptide in homogenates of bovine retina. Localization of this polypeptide in bovine retina by indirect immunofluorescence demonstrates that it is expressed only in cone outer segments. Antibodies to specific sequences found only in the rod transducin alpha subunit recognize a polypeptide localized only in the rod outer segment. Therefore, bovine rod and cone cells each express structurally related yet significantly different forms of transducin.     

Phagocytosis in the retinal pigment epithelium of the RCS rat

The retinal pigment epithelium of RCS rats, previously thought not to phagocytize photoreceptor outer segments, exhibited a peak of phagocytosis in vivo when animals were kept under conditions of cyclic lighting (12 hours of darkness and 12 hours of light). The peak occurred at 1 hour after the onset of light, with maximum and minimum levels of phagocytosis averaging about 5 percent of that found in the pigment epithelium of Osborn-Mendel rats used as a control. Eyecups that were obtained from Osborn-Mendel rats and maintained for up to 3 hours in organ culture demonstrated levels of phagocytosis that were sevenfold greater than those of unincubated controls. Likewise a tenfold increase occurred in incubated as opposed to unicubated RCS eyes, raising the possibility that phagocytosis could be experimentally stimulated in vivo.     

Phagocytosis of light- and dark-adapted rod outer segments by cultured pigment epithelium

Pigment epithelial cells in culture retain their ability to phagocytize rod outer segments. These cells phagocytize rod outer segments isolated from light-adapted rats, or from dark-adapted rats killed after the time at which the lights would normally be turned on. However, they phagocytize for fewer rod outer segments prepared in the dark from the retinas of rats killed before the onset of the normal light cycle. Phagocytosis of dark rod outer segments is variable, but that of light outer segments is reproducible. It is postulated that the effect of light is to synchronize the chemical events that occur at the surface of the rods to prepare them for phagocytosis. These processes also occur in the dark, but more slowly and irregularly than in the light.     

Phototropism in Phycomyces as investigated by focused laser radiation

The phototropic response and distribution of photopigment in the sporangiophore of Phycomyces was investigated with a microillumination pattern. The data support the hypothesis that phototropism results from greater stimulation of growth in regions of more intense illumination and indicate that the photoreceptor extends to the outer wall of the sporangiophore.     

Topology of the outer segment membranes of retinal rods and cones revealed by a fluorescent probe

When N,N'-didansyl cystine binds to the cell membranes of vertebrate rods and cones its fluorescence efficiency increases about 20-fold. The entire outer segments of living cones become brilliantly fluorescent. Stained live rods, as well as most freshly detached outer segments, are only weakly fluorescent, but they become brightly fluorescent within a few seconds if their plasma membranes are osmotically ruptured. The difference in staining of rod and cones suggests that disk membranes of rods are not continuous with the plasma membranes are osmotically ruptured. The difference in staining of rod and cones plasma membrane on outer segments of photoreceptors in electrophysiological and biochemical experiments, and to study the infolding pattern of rod and cone disks.     

Cones of living amphibian eye: selective staining

The outer segments of cones are selectively stained by the chlortriazinyl dye Procion Yellow injected into the vitreous humor. Since the dye does not cross nerve cell membranes, the selective staining of cones is further evidence for structural differences between rod and cone outer segments. Specifically, it is believed that cone saccules are open to extracellular space whereas rod saccules are not.     

Membrane origin of the fast photovoltage of squid retina

When a bright light flash is absorbed by a small region in the outer segments of squid photoreceptors fixed in glutaraldehyde, a brief pulse of membrane current flows locally. The passive spreading of this current along the outer segments produces the photochemical component of the "early receptor potential." The source of the current lies electrically in parallel with the cell membranes and perhaps is located within them. Fixation with glutaraldehyde apparently does not reduce the resistance of the cell membrane to less than 5 percent of its value in live cells.     

Ocular hazard from picosecond pulses of Nd: YAG laser radiation

Seven rhesus monkeys (14 eyes) were exposed to 1064-nanometer radiation in single pulses of 25 to 35 picoseconds fromn a mode-locked Nd: YA G laser. Threshold injury resulted from single pulses with a mnean energy of 13 +/- 3 mnicrojoules. Electron microscopy of the retina revealed that damnage was highly localized in the photoreceptor and pigmented epithelial cells at the oluter retina. Membrane disruption, distorted outer segmtzents, and abnormnal melanin granules resembling fetal premelanosomnies were observed.     

Defective phagocytosis of isolated rod outer segments by RCS rat retinal pigment epithelium in culture

Retinal pigment epithelium cultured from normal rats phagocytizes large amounts of rod outer segment fragments isolated from normal rats and from RCS rats with inherited retinal degeneration. Cultured RCS rat pigment epithelium rarely ingests outer segment material, although the cells extend cellular processes around fragments of either type. Both normal and RCS pigment epithelium phagocytize polystyrene spheres. This demonstrates that RCS rat pigment epithelial cells contain a defect in the mechanism for phagocytizing outer segments.     

Rescue of photoreceptor degeneration in rhodopsin-null Drosophila mutants by activated Rac1

Rhodopsin is essential for photoreceptor morphogenesis; photoreceptors lacking rhodopsin degenerate in humans, mice, and Drosophila. Here we report that transgenic expression of a dominant-active Drosophila Rho guanosine triphosphatase, Drac1, rescued photoreceptor morphogenesis in rhodopsin-null mutants; expression of dominant-negative Drac1 resulted in a phenotype similar to that seen in rhodopsin-null mutants. Drac1 was localized in a specialization of the photoreceptor cortical actin cytoskeleton, which was lost in rhodopsin-null mutants. Thus, rhodopsin appears to organize the actin cytoskeleton through Drac1, contributing a structural support essential for photoreceptor morphogenesis.     

Wave-guide modes in retinal receptors

Retinal receptor wave-guide modal patterns have been photographed. The inner and outer segments of the rod and cone receptors of albino rats, rhesus macaque monkeys, and human beings have been studied. The wave-guide modal pattern is believed to be the normal form of energy transfer in these parts of the retinal receptors in these species.     

Retinal S antigen identified as the 48K protein regulating light-dependent phosphodiesterase in rods

Retinal S antigen chromatographically purified from whole retina, induces experimental autoimmune uveoretinitis in laboratory animals. The 48K protein, a soluble protein found in rod outer segments, is purified through its specific binding to photoexcited rhodopsin and is involved in the quenching of light-induced guanosine 3',5'-monophosphate-phosphodiesterase activity. Biochemical, immunological, functional, and pathological tests showed that retinal S antigen and the 48K protein are identical.     

Lateral diffusion of visual pigment in photorecptor disk membranes

Visual pigment molecules are found to move transversely, but not longitudinally, in both rod and cone outer segments of mud puppy and frog. This is consistent with the idea that they are immersed in a two-dimensional fluid disk membrane. The diffusion coefficient for the motion is about 5 x 10(-9) square centimeters per second at 20 degrees C, corresponding to a root-mean-square molecular displacement of 0.3 micrometer in 1 second.     

Ellipsosomes: organelles containing a cytochrome-like pigment in the retinal cones of certain fishes

Ellipsosomes are dense spherical bodies containing a very large concentration of a heme pigment spectroscopically resembling pure cytochrome c. They are located at the outer ends of the inner segments of the cones of certain fishes. Although, superficially, they resemble the similarly located oil droplets in the cones of birds and reptiles, their ultrastructure and staining properties resemble those of the neighboring mitochondria. However, like the oil droplets, they may serve as intracellular color filters.     

Divalent cations directly affect the conductance of excised patches of rod photoreceptor membrane

Phototransduction in rod cells is likely to involve an intracellular messenger system that links the absorption of light by rhodopsin to a change in membrane conductance. The direct effect of guanosine 3',5'-monophosphate (cGMP) on excised patches of rod outer segment membrane strongly supports a role for cGMP as an intracellular messenger in phototransduction. It is reported here that magnesium and calcium directly affect the conductance of excised patches of rod membrane in the absence of cGMP and that magnesium, applied to intact rod cells, blocks a component of the cellular light response. The divalent cation-suppressed conductance in excised patches showed outward rectification and cation-selective permeability resembling those of the light-suppressed conductance measured from the intact rod cell. The divalent cation-suppressed conductance was partly blocked by a concentration of the pharmacological agent L-cis-diltiazem that blocked all of the cGMP-activated conductance. Divalent cations may act, together with cGMP, as an intracellular messenger system that mediates the light response of the rod photoreceptor cell.     

Visual pigment fluorescence

The fluorescence of cattle rod outer segments (dried) and of rhodopsin in solution lies in the range of 575 to 600 millimicrons with a quantum efficiency of 0.005 if excitation is in the visible band near 500 millimicrons. The emission is abolished by bleaching at -196 degrees C but can be reversibly regenerated by irradiation with light of longer wavelength (600 millimicrons). This behavior reflects the known interconversion of rhodopsin to prelumirhodopsin at this temperature.     

Cyclic nucleotide phosphodiesterase: high activity in a mammalian photoreceptor

Purified outer segments of bovine rods exhibit phosphodiesterase activity against adenosine and guanosine cyclic 3',5'-monophosphates (cyclic AMP and cyclic GMP). The enzyme hydrolyzed cyclic GMP more rapidly than cyclic AMP at low substrate concentrations. The presence of high phosphodiesterase activity in this highly specialized organelle suggests that this enzyme may function in control of cyclic nucleotide concentration during visual excitation or adaptation.