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PubMed2002

Rhodopsin phosphorylation: from terminating single photon responses to photoreceptor dark adaptation.

Rhodopsin phosphorylation has provided one of the first examples of the ubiquitous regulatory pattern of specific kinases downregulating the activity of G-protein-coupled receptors. However, only recently have studies in living animals allowed us to consider the role of rhodopsin phosphorylation in a broader spectrum of visual functions, ranging from the ability of rods to generate reproducible electrical responses to their ability to adapt to darkness after substantial light exposures.

Animals↗
PubMed2002

Distribution of two splice variants of the glutamate transporter GLT1 in the retinas of humans, monkeys, rabbits, rats, cats, and chickens.

Antibodies have been generated against two carboxyl-terminal splice variants of the glutamate transporter GLT1, namely, the originally described version of GLT1 and GLT1-B, and labelling has been examined in multiple species, including chickens and humans. Although strong specific labelling was observed in each species, divergent patterns of expression were noted. Moreover, each antibody was sensitive to the phosphorylation state of the appropriate protein, because chemical removal of phosphates using alkaline phosphatase revealed a broader range of labelled elements in most cases. In general, GLT1-B was present in cone photoreceptors and in rod and cone bipolar cells in the retinas of rabbits, rats, and cats. In the cone-dominated retinas of chickens and in marmosets, GLT1-B was associated only with cone photoreceptors, whereas, in macaque and human retinas, GLT1-B was associated with bipolar cells and terminals of photoreceptors. In some species, such as cats, GLT-B was also present in horizontal cells. By contrast, GLT1 distribution varied. GLT1 was associated with amacrine cells in chickens, rats, cats, and rabbits and with bipolar cells in marmosets and macaques. In the rat retina, rod photoreceptor terminals also contained GLT1, but this was evident only in enzymatically dephosphorylated tissues. We conclude that the two variants of GLT1 are present in all species examined but are differentially distributed in a species-specific manner. Moreover, each cell type generally expresses only one splice variant of GLT1.

Amacrine Cells↗
PubMedDate not supplied

Subcellular localization of phosducin in rod photoreceptors.

Phosducin (Pd) is a 28-kD phosphoprotein whose expression in retina appears limited to photoreceptor cells. Pd binds to the beta,gamma subunits of transducin (Gt). Their binding affinity is markedly diminished by Pd phosphorylation. While Pd has long been regarded as a candidate for the regulation of Gt, the molecular details of Pd function remain unclear. This gap in understanding is due in part to a lack of precise information concerning the total amount and subcellular localization of rod Pd. While earlier studies suggested that Pd was a rod outer segment (ROS) protein, recent findings have demonstrated that Pd is distributed throughout the rod. In this report, the subcellular distribution and amounts of rat Pd are quantified with immunogold electron microscopy. After light or dark adaptation, retinal tissues were fixed in situ and prepared for ultrathin sectioning and immunogold labeling. Pd concentrations were analyzed over the entire length of the rod. The highest Pd labeling densities were found in the rod synapse. Less intense Pd staining was observed in the ellipsoid and myoid regions, while minimal labeling densities were found in the ROS and the rod nucleus. In contrast with rod Gt, no evidence was found for light-dependent movement of Pd between inner and outer segments. There is a relative paucity of Pd in the ROS as compared with the large amounts of Gt found there. This does not support the earlier idea that Pd could modulate Gt activity by controlling its concentration. On the other hand, the presence of Pd in the nucleus is consistent with its possible role as a regulator of transcription. The functions of Pd in the ellipsoid and myoid regions remain unclear. The highest concentration of Pd was found at the rod synapse, consistent with a suggested role for Pd in the regulation of synaptic function.

Animals↗
PubMedDate not supplied

Response linearity and kinetics of the cat retina: the bipolar cell component of the dark-adapted electroretinogram.

The electroretinogram (ERG) of the dark-adapted cat eye in response to brief ganzfeld flashes of a wide range of intensities was recorded after intravitreal injection of n-methyl DL aspartate (NMDLA, cumulative intravitreal concentration of 1.3-3.9 mM) to suppress inner-retinal components, and after intravitreal DL or L-2-amino-4-phosphonobutyric acid (DL-APB, 1-3 mM; L-APB, 1.2 mM) and 6-cyano-7-nitroquinoxaline-2,3 dione (CNQX, 40-60 microM), to suppress all post-receptoral neuronal responses. Rod PII, the ERG component arising from rod bipolar cells, was derived by subtracting records obtained after APB and CNQX from post-NMDLA records. When we measured the derived response at fixed times after the stimulus, we found that PII initially increased in proportion to stimulus intensity without any sign of a threshold. The leading edge of PII at early times after the stimulus, when the response was still small, was well described by V(t) = kI(t-td)5 where k is a constant, I is the intensity of the stimulus, and td is a brief delay of about 3 ms. Correspondingly, the time for the response to rise to an arbitrary small criterion voltage Vcrit was adequately fitted by tcrit = td + (Vcrit/kI)1/5. The time course of the leading edge of the PII response can be interpreted to indicate that the mechanism generating PII introduces three stages of temporal integration in addition to the three stages that are provided by the mechanism of the rod photoreceptors. This finding is consistent with the operation within the rod bipolar cell of a G-protein cascade similar to that in the rods.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2006

Cyclic nucleotide-gated ion channels in rod photoreceptors are protected from retinoid inhibition.

In vertebrate rods, photoisomerization of the 11-cis retinal chromophore of rhodopsin to the all-trans conformation initiates a biochemical cascade that closes cGMP-gated channels and hyperpolarizes the cell. All-trans retinal is reduced to retinol and then removed to the pigment epithelium. The pigment epithelium supplies fresh 11-cis retinal to regenerate rhodopsin. The recent discovery that tens of nanomolar retinal inhibits cloned cGMP-gated channels at low [cGMP] raised the question of whether retinoid traffic across the plasma membrane of the rod might participate in the signaling of light. Native channels in excised patches from rods were very sensitive to retinoid inhibition. Perfusion of intact rods with exogenous 9- or 11-cis retinal closed cGMP-gated channels but required higher than expected concentrations. Channels reopened after perfusing the rod with cellular retinoid binding protein II. PDE activity, flash response kinetics, and relative sensitivity were unchanged, ruling out pharmacological activation of the phototransduction cascade. Bleaching of rhodopsin to create all-trans retinal and retinol inside the rod did not produce any measurable channel inhibition. Exposure of a bleached rod to 9- or 11-cis retinal did not elicit channel inhibition during the period of rhodopsin regeneration. Microspectrophotometric measurements showed that exogenous 9- or 11-cis retinal rapidly cross the plasma membrane of bleached rods and regenerate their rhodopsin. Although dark-adapted rods could also take up large quantities of 9-cis retinal, which they converted to retinol, the time course was slow. Apparently cGMP-gated channels in intact rods are protected from the inhibitory effects of retinoids that cross the plasma membrane by a large-capacity buffer. Opsin, with its chromophore binding pocket occupied (rhodopsin) or vacant, may be an important component. Exceptionally high retinoid levels, e.g., associated with some retinal degenerations, could overcome the buffer, however, and impair sensitivity or delay the recovery after exposure to bright light.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Identification of the alpha-subunits of rod and cone transducin in chicken photoreceptor cells.

The chicken retina has several types of cone photoreceptor cells, each of which contains a visual pigment, chicken red (iodopsin), green, blue or violet. Although biochemical and photochemical properties of these cone pigments have been well characterized, no information is available about the chicken photoreceptor G-protein, transducin, which couples with the visual pigment to convert a photon signal into a cellular response. To identify alpha-subunits of chicken rod and cone transducins (Tr alpha and Tc alpha, respectively), we produced two site-directed antibodies which discriminate between bovine Tr alpha and Tc alpha. Immunohistochemical studies on chicken retinas revealed that the antibody against bovine Tr alpha specifically stained the rod outer segments. On the other hand, the antibody against bovine Tc alpha uniformly stained the outer segments of the double cones and all types of single cones, while the single cones were immunohistochemically classified into three types by using a combination of antibodies against bovine rhodopsin and chicken iodopsin. Immuno-blot analyses demonstrated that the antibody against Tc alpha recognized a single band of chicken photoreceptor protein, whose molecular weight (42,000) was in good agreement with that of bovine Tc alpha (41,000). The antibody against Tr alpha recognized a protein having the same molecular weight as that of bovine Tr alpha (39,000). These observations strongly suggested that all types of chicken cone cells have a single common Tc alpha (42 kDa) structurally related to bovine Tc alpha, though each cone cell type has a distinct visual pigment.

Animals↗
PubMed1999

Rod photoreceptors in infant rats with a history of oxygen exposure.

PURPOSE: To study in an infant rat model of retinopathy of prematurity, the rod photoreceptors, which are known to have attenuated photoresponses. METHODS: Rhodopsin was extracted from whole retinas, the thickness of the rod outer segment (ROS) layer was measured, large phagosomes were counted, and the ROS ultrastructure was examined in the retinas of oxygen-exposed and control rats, ages 13 and 18 days. Rhodopsin absorbances in the ROS were measured by microspectrophotometry at age 20 days. RESULTS: The rhodopsin content did not differ significantly between the oxygen-exposed and control rats at either 13 or 18 days. The thickness of the ROS layer was equal in 13-day-old oxygen-exposed and control rats; however, at 18 days, the ROS layer was significantly thinner in the oxygen-exposed rats than in the control rats. The number of phagosomes did not vary significantly among the oxygen-exposed and control groups. Opsin immunoreactivity was seen only in the ROS layer in oxygen-exposed and control rats. The ROS were disorganized in oxygen-exposed rats. The rhodopsin absorbances of the oxygen-exposed ROS were significantly more variable and higher than in the control rats. CONCLUSIONS: Attenuation of the rod photoresponse parameters does not result simply from shortening of the outer segments and consequent low rhodopsin content. Rather, the structure of the outer segments is altered. A fault in the synthesis of the outer segments, rather than disposal of outer segment discs, is suspected.

Animals↗
PubMed2004

Electromagnetic field of extremely low frequency decreased adenylate kinase activity in retinal rod outer segment membranes.

Adenylate kinase activity in rod outer segment membranes of bovine retina decreased of about 55% when exposed to an extremely low frequency electromagnetic field of 75 Hz and 250 microT. The effect was independent of the time of permanence in the field. Negligible effects of the field were found on the enzymatic activity of a soluble isoform of adenylate kinase or of rod outer segment membranes solubilized with Triton, suggesting the importance of the membrane in determining the conditions of the enzyme inactivation.

Adenosine Triphosphate↗
PubMed1999

Normal and mutant rhodopsin activation measured with the early receptor current in a unicellular expression system.

The early receptor current (ERC) represents molecular charge movement during rhodopsin conformational dynamics. To determine whether this time-resolved assay can probe various aspects of structure-function relationships in rhodopsin, we first measured properties of expressed normal human rhodopsin with ERC recordings. These studies were conducted in single fused giant cells containing on the order of a picogram of regenerated pigment. The action spectrum of the ERC of normal human opsin regenerated with 11-cis-retinal was fit by the human rhodopsin absorbance spectrum. Successive flashes extinguished ERC signals consistent with bleaching of a rhodopsin photopigment with a normal range of photosensitivity. ERC signals followed the univariance principle since millisecond-order relaxation kinetics were independent of the wavelength of the flash stimulus. After signal extinction, dark adaptation without added 11-cis-retinal resulted in spontaneous pigment regeneration from an intracellular store of chromophore remaining from earlier loading. After the ERC was extinguished, 350-nm flashes overlapping metarhodopsin-II absorption promoted immediate recovery of ERC charge motions identified by subsequent 500-nm flashes. Small inverted R(2) signals were seen in response to some 350-nm flashes. These results indicate that the ERC can be photoregenerated from the metarhodopsin-II state. Regeneration with 9-cis-retinal permits recording of ERC signals consistent with flash activation of isorhodopsin. We initiated structure-function studies by measuring ERC signals in cells expressing the D83N and E134Q mutant human rhodopsin pigments. D83N ERCs were simplified in comparison with normal rhodopsin, while E134Q ERCs had only the early phase of charge motion. This study demonstrates that properties of normal rhodopsin can be accurately measured with the ERC assay and that a structure-function investigation of rapid activation processes in analogue and mutant visual pigments is feasible in a live unicellular environment.

Cell Line↗
PubMed2005

Spare the rod and spoil the eye.

This review presents a new unified view of the pathogenesis of three common causes of acquired retinal degenerative disease-diabetic retinopathy, age related macular degeneration, and retinopathy of prematurity. In these three conditions, angiogenesis has a predominant role in the development of sight threatening pathology. Angiogenesis is controlled by among other factors the expression of vascular endothelial growth factor (VEGF), which in turn is regulated by absolute and relative lack of oxygen. The severe pathological manifestations of these three conditions are not part of a general underlying disease process because they are peculiar to the eye, and the profound hypoxia that develops in normal retina during dark adaptation (rod driven hypoxia) is an adequate and elegant additional factor to explain their pathogenesis. A large number of experimental reports support this conclusion, although rod driven anoxia is not generally considered as a causal factor in ocular disease. However, the hypothesis can be critically tested, and also suggests novel methods of treatment and prevention of these conditions that may be simpler and more inexpensive than current therapies and that have a smaller potential for adverse effects.

Animals↗
PubMed1995

GABA- and glycine-activated currents in the rod bipolar cell of the rabbit retina.

1. Voltage- and ligand-gated currents were recorded from solitary rabbit rod bipolar cells using the whole cell patch-clamp technique. The rod bipolar cell forms a single, stereotypical physiological and morphological class of cells that was easily identified from other neurons and support cells after enzymatic and mechanical dissociation from isolated retina. Protein kinase C immunoreactivity confirmed the validity of using a purely morphological identification of this cell type. 2. Voltage steps in 15-mV increments from a holding potential of -45 mV elicited a large outward current activated near -30 mV. These voltage-gated currents were eliminated by using equimolar substitutions of Cs+ and tetraethylammonium+ for K+ in the pipette, indicating that they represent a mixture of K+ currents. 3. The putative inhibitory neurotransmitters gamma-aminobutyric acid (GABA) and glycine activated inward Cl- currents when pressure-applied from pipettes placed near the axon terminals of rod bipolar cells, which were voltage-clamped at -45 mV. With changes in intracellular or extracellular Cl- concentration, the reversal potential of these ligand-gated currents changed as predicted by the Nernst equation for Cl- activity. The dose-response curves for GABA and glycine were sigmoidal with saturating concentrations of 100 and 300 microM, respectively. 4. GABA-activated currents were 1) reversibly reduced by the allosteric inhibitor picrotoxin and the competitive antagonist bicuculline; 2) potentiated by the benzodiazepine diazepam and the barbiturate barbital sodium; and 3) indistinguishable from muscimol-activated currents. There was no response to the GABAB agonist baclofen. Collectively, these data strongly suggest that the GABA-activated currents in rabbit rod bipolar cells are mediated by the GABAA receptor. This is similar to the GABA-activated currents in other mammalian rod bipolar cells. 5. Application of the conformationally restricted GABA analogue cis-4-aminocrotonic acid (CACA) failed to elicit a response, whereas the conformationally extended GABA analogue trans-4-aminocrotonic acid (TACA) elicited a response similar to that of GABA. Although bicuculline appeared to suppress the GABA-activated current slightly more than the TACA-activated current (not significant using Student's t-distribution), GABA- and TACA-activated currents were equally suppressed by picrotoxin and equally enhanced by diazepam and barbital sodium. These data, coupled with the inefficacy of CACA, argue against the existence of a GABAC-type channel in the rod bipolar cell of the rabbit and suggest that GABA and TACA were activating the same GABAA receptor-channel complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

Are there ionotropic glutamate receptors on the rod bipolar cell of the mouse retina?

There is some evidence that the mammalian rod bipolar cell expresses ionotropic glutamate receptors. This is surprising in light of the strong evidence that the glutamate released by the rod photoreceptor acts upon a metabotropic glutamate receptor-mGluR6-present in the dendrites of the rod bipolar cell. To reexamine the issue of which glutamate receptor subunits may be present on the rod bipolar cell, an immunohistochemical study of acutely dissociated retinal cells was undertaken. Two monoclonal antibodies provided some evidence that GluR2 and/or GluR4, as well as NMDAR1 subunit, are present on the rod bipolar cell. A monoclonal antibody directed against the N-terminus of GluR2 labeled the rod bipolar cells, but two antisera directed against the C-terminus of the same subunit did not. One possible explanation for this discrepancy could be that the rare splice variant GluR2-long, which is endowed with a different C-terminus, could be expressed by the rod bipolar cell. To explore this possibility, RT-PCR was used to amplify the transcripts encoding GluR2 in the neural retina. This revealed that GluR2-long transcripts, with the flop exon, are present.

Animals↗
PubMed2001

Comprehensive analysis of photoreceptor gene expression and the identification of candidate retinal disease genes.

To identify the full set of genes expressed by mammalian rods, we conducted serial analysis of gene expression (SAGE) by using libraries generated from mature and developing mouse retina. We identified 264 uncharacterized genes that were specific to or highly enriched in rods. Nearly half of all cloned human retinal disease genes are selectively expressed in rod photoreceptors. In silico mapping of the human orthologs of genes identified in our screen revealed that 86 map within intervals containing uncloned retinal disease genes, representing 37 different loci. We expect these data will allow identification of many disease genes, and that this approach may be useful for cloning genes involved in classes of disease where cell type-specific expression of disease genes is observed.

Animals↗
PubMed1997

Identification and characterization of a conserved family of protein serine/threonine phosphatases homologous to Drosophila retinal degeneration C.

The Drosophila retinal degeneration C (rdgC) gene encodes an unusual protein serine/threonine phosphatase in that it contains at least two EF-hand motifs at its carboxy terminus. By a combination of large-scale sequencing of human retina cDNA clones and searches of expressed sequence tag and genomic DNA databases, we have identified two sequences in mammals [Protein Phosphatase with EF-hands-1 and 2 (PPEF-1 and PPEF-2)] and one in Caenorhabditis elegans (PPEF) that closely resemble rdgC. In the adult, PPEF-2 is expressed specifically in retinal rod photoreceptors and the pineal. In the retina, several isoforms of PPEF-2 are predicted to arise from differential splicing. The isoform that most closely resembles rdgC is localized to rod inner segments. Together with the recently described localization of PPEF-1 transcripts to primary somatosensory neurons and inner ear cells in the developing mouse, these data suggest that the PPEF family of protein serine/threonine phosphatases plays a specific and conserved role in diverse sensory neurons.

Alternative Splicing↗
PubMed2004

Quantification of the cytoplasmic spaces of living cells with EGFP reveals arrestin-EGFP to be in disequilibrium in dark adapted rod photoreceptors.

The hypothesis is tested that enhanced green fluorescent protein (EGFP) can be used to quantify the aqueous spaces of living cells, using as a model transgenic Xenopus rods. Consistent with the hypothesis, regions of rods having structures that exclude EGFP, such as the mitochondrial-rich ellipsoid and the outer segments, have highly reduced EGFP fluorescence. Over a 300-fold range of expression the average EGFP concentration in the outer segment was approximately half that in the most intensely fluorescent regions of the inner segment, in quantitative agreement with prior X-ray diffraction estimates of outer segment cytoplasmic volume. In contrast, the fluorescence of soluble arrestin-EGFP fusion protein in the dark adapted rod outer segment was approximately threefold lower than predicted by the EGFP distribution, establishing that the fusion protein is not equilibrated with the cytoplasm. Arrestin-EGFP mass was conserved during a large-scale, light-driven redistribution in which approximately 40% of the protein in the inner segment moved to the outer segment in less than 30 minutes.

Animals↗
PubMed1993

Expression of recoverin mRNA in the human retina: localization by in situ hybridization.

The localization of mRNA encoding recoverin in the human retina was examined by in situ hybridization. The riboprobe utilized in this study was transcribed from a cDNA clone containing the complete coding region of human recoverin. The riboprobe hybridized extensively with the photoreceptor inner segments, and the outer nuclear layer. Specific hybridization was also seen in a subpopulation of cells in the inner nuclear layer, and occasionally in cells of the ganglion cell layer (GCL). Recoverin-like immunoreactivity was observed in all photoreceptors, and in a subpopulation of bipolar cells. Very robust immunolabeling was observed in a small population of cells in the GCL. This study suggests that recoverin, or a recoverin-like protein, is produced by at least three different cell types in the human retina: photoreceptors, bipolars and rare cells in the ganglion cell layer.

Adult↗
PubMed2002

GABA(C) receptors modulate the rod-driven ERG b-wave of the skate retina.

Studies characterizing the types of GABA receptors present on cells isolated from the skate retina have allowed us to develop a working model of possible GABA interactions at the level of the outer plexiform layer (OPL). Earlier studies have shown an electrogenic GABA transport mechanism in horizontal cells presents a source of GABA in the OPL which could modulate feedback onto photoreceptors. GABA(A) receptors on Müller cells, or GABA(A) and/or GABA(C) receptors on bipolar cells. This model has been used for the interpretation of results of experiments in this study designed to test the role these interactions may exert on the electroretinogram (ERG). Simultaneous intracellular recording of the horizontal cell response (the S-potential) was used to monitor effects on photoreceptor transmitter release which would be altered if GABAergic photoreceptor feedback mechanisms were involved. Picorotoxin (50 microM), a chloride channel blocker which suppresses the responses of both GABA(A)Rs and GABA(C)Rs, reduced the ON (b-wave) component of the ERG substantially. Simultaneous intracellular horizontal cell recordings, however, showed no effect on their light-evoked response, suggesting that photoreceptor feedback is not involved in the picrotoxin effect on the ERG. On the other hand, even 100 microM bicuculline, a GABA(A)R antagonist produced no change in either the ERG or the horizontal cell response. This observation leads to the conclusion that the GABAARs on Miller cells and bipolar cells are not involved. Thus, there remains a distinct possibility that the ERG changes produced by picrotoxin are due to its ability to block the GABA(C)Rs on retinal bipolar cells.

Animals↗
PubMed2000

Peripherin/rds influences membrane vesicle morphology. Implications for retinopathies.

Peripherin/rds is an integral membrane glycoprotein found in the rim regions of vertebrate photoreceptor cell discs. Natural mutations of the encoding gene result in degenerative retinal disorders, such as retinitis pigmentosa. The retinal degeneration slow (rds) phenotype, observed in mice, is considered to be an appropriate model for peripherin/rds-mediated retinitis pigmentosa. Associated abnormalities in the outer segment of photoreceptor cells have implicated peripherin/rds in some aspect of disc morphology, yet it remains unclear whether such morphological effects are the cause or the result of this condition. Here we present the first direct evidence to support a role for peripherin/rds in maintaining the flattened vesicle morphology characteristic of photoreceptor outer segments. In vitro expression yields a 36-kDa immunoreactive species, which is inserted into membranes and undergoes N-glycosylation, inter- and intramolecular disulfide bonding, and dimerization. Electron microscopy reveals that peripherin/rds flattens microsomal vesicles. This effect appears to be dependent on disulfide bond formation but not N-glycosylation. The inability of two pathogenic peripherin/rds mutants (P216L and C165Y) to flatten membrane vesicles implicates such mutations as the primary cause of the retinal degeneration observed in retinitis pigmentosa.

Animals↗