Physiological and Microfluorometric Studies of Reduction and Clearance of Retinal in Bleached Rod Photoreceptors

The visual cycle comprises a sequence of reactions that regenerate the visual pigment in photoreceptors during dark adaptation, starting with the reduction of all-trans retinal to all-trans retinol and its clearance from photoreceptors. We have followed the reduction of retinal and clearance of retinol within bleached outer segments of red rods isolated from salamander retina by measuring its intrinsic fluorescence. Following exposure to a bright light (bleach), increasing fluorescence intensity was observed to propagate along the outer segments in a direction from the proximal region adjacent to the inner segment toward the distal tip. Peak retinol fluorescence was achieved after ∼30 min, after which it declined very slowly. Clearance of retinol fluorescence is considerably accelerated by the presence of the exogenous lipophilic substances IRBP (interphotoreceptor retinoid binding protein) and serum albumin. We have used simultaneous fluorometric and electrophysiological measurements to compare the rate of reduction of all-trans retinal to all-trans retinol to the rate of recovery of flash response amplitude in these cells in the presence and absence of IRBP. We find that flash response recovery in rods is modestly accelerated in the presence of extracellular IRBP. These results suggest such substances may participate in the clearance of retinoids from rod photoreceptors, and that this clearance, at least in rods, may facilitate dark adaptation by accelerating the clearance of photoproducts of bleaching.

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Turning Cones Off: the Role of the 9-Methyl Group of Retinal in Red Cones

The Journal of General Physiology ◽

10.1085/jgp.200609630 ◽

2006 ◽

Vol 128 (6) ◽

pp. 671-685 ◽

Cited By ~ 8

Author(s):

Maureen E. Estevez ◽

Petri Ala-Laurila ◽

Rosalie K. Crouch ◽

M. Carter Cornwall

Keyword(s):

Visual Pigment ◽

Bright Light ◽

Rapid Rate ◽

Methyl Group ◽

Cone Opsin ◽

Rate Of Recovery ◽

Kinetics Of ◽

Flash Response

Our ability to see in bright light depends critically on the rapid rate at which cone photoreceptors detect and adapt to changes in illumination. This is achieved, in part, by their rapid response termination. In this study, we investigate the hypothesis that this rapid termination of the response in red cones is dependent on interactions between the 9-methyl group of retinal and red cone opsin, which are required for timely metarhodopsin (Meta) II decay. We used single-cell electrical recordings of flash responses to assess the kinetics of response termination and to calculate guanylyl cyclase (GC) rates in salamander red cones containing native visual pigment as well as visual pigment regenerated with 11-cis 9-demethyl retinal, an analogue of retinal in which the 9-methyl group is missing. After exposure to bright light that photoactivated more than ∼0.2% of the pigment, red cones containing the analogue pigment had a slower recovery of both flash response amplitudes and GC rates (up to 10 times slower at high bleaches) than red cones containing 11-cis retinal. This finding is consistent with previously published biochemical data demonstrating that red cone opsin regenerated in vitro with 11-cis 9-demethyl retinal exhibited prolonged activation as a result of slowed Meta II decay. Our results suggest that two different mechanisms regulate the recovery of responsiveness in red cones after exposure to light. We propose a model in which the response recovery in red cones can be regulated (particularly at high light intensities) by the Meta II decay rate if that rate has been inhibited. In red cones, the interaction of the 9-methyl group of retinal with opsin promotes efficient Meta II decay and, thus, the rapid rate of recovery.

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Vitamin A activates rhodopsin and sensitizes it to ultraviolet light

Visual Neuroscience ◽

10.1017/s0952523811000423 ◽

2011 ◽

Vol 28 (6) ◽

pp. 485-497 ◽

Cited By ~ 2

Author(s):

SADAHARU MIYAZONO ◽

TOMOKI ISAYAMA ◽

FRANÇOIS C. DELORI ◽

CLINT L. MAKINO

Keyword(s):

Vitamin A ◽

Uv Light ◽

Electrical Response ◽

Bright Light ◽

Binding Pocket ◽

Outer Segments ◽

Circulating Current ◽

Retinal Chromophore ◽

Flash Response

AbstractThe visual pigment, rhodopsin, consists of opsin protein with 11-cis retinal chromophore, covalently bound. Light activates rhodopsin by isomerizing the chromophore to the all-trans conformation. The activated rhodopsin sets in motion a biochemical cascade that evokes an electrical response by the photoreceptor. All-trans retinal is eventually released from the opsin and reduced to vitamin A. Rod and cone photoreceptors contain vast amounts of rhodopsin, so after exposure to bright light, the concentration of vitamin A can reach relatively high levels within their outer segments. Since a retinal analog, β-ionone, is capable of activating some types of visual pigments, we tested whether vitamin A might produce a similar effect. In single-cell recordings from isolated dark-adapted salamander green-sensitive rods, exogenously applied vitamin A decreased circulating current and flash sensitivity and accelerated flash response kinetics. These changes resembled those produced by exposure of rods to steady light. Microspectrophotometric measurements showed that vitamin A accumulated in the outer segments and binding of vitamin A to rhodopsin was confirmed in in vitro assays. In addition, vitamin A improved the sensitivity of photoreceptors to ultraviolet (UV) light. Apparently, the energy of a UV photon absorbed by vitamin A transferred by a radiationless process to the 11-cis retinal chromophore of rhodopsin, which subsequently isomerized. Therefore, our results suggest that vitamin A binds to rhodopsin at an allosteric binding site distinct from the chromophore binding pocket for 11-cis retinal to activate the rhodopsin, and that it serves as a sensitizing chromophore for UV light.

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Movement of retinal along cone and rod photoreceptors

Visual Neuroscience ◽

10.1017/s0952523800001735 ◽

1994 ◽

Vol 11 (2) ◽

pp. 389-399 ◽

Cited By ~ 29

Author(s):

Jing Jin ◽

Gregor J. Jones ◽

M. Carter Cornwall

Keyword(s):

Cell Body ◽

Dark Adaptation ◽

Visual Pigment ◽

Outer Segment ◽

Light Adaptation ◽

Rod Photoreceptors ◽

Rod Outer Segment ◽

Rods And Cones ◽

Outer Segments ◽

Rod Cell

AbstractSingle isolated photoreceptors can be taken through a visual cycle of light adaptation by bleaching visual pigment, followed by dark adaptation when supplied with 11–cis retinal. Light adaptation after bleaching is manifested by faster response kinetics and a permanent reduction in sensitivity to light flashes, presumed to be due to the presence of bleached visual pigment. The recovery of flash sensitivity during dark adaptation is assumed to be due to regeneration of visual pigment to pre-bleach levels. In previous work, the outer segments of bleached, light-adapted cells were exposed to 11–cis retinal. In the present work, the cell bodies of bleached photoreceptors were exposed. We report a marked difference between rods and cones. Bleached cones recover sensitivity when their cell bodies are exposed to 11–cis retinal. Bleached rods do not. These results imply that retinal can move freely along the cone photoreceptor, but retinal either is not taken up by the rod cell body or retinal cannot move from the rod cell body to the rod outer segment. The free transfer of retinal along cone but not along rod photoreceptors could explain why, during dark adaptation in the retina, cones have access to a store of 11–cis retinal which is not available to rods. Additional experiments investigated the movement of retinal along bleached rod outer segments. The results indicate that retinal can move along the rod outer segment, but that this movement is slow, occurring at about the same rate as the regeneration of visual pigment.

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The retina visual cycle is driven by cis retinol oxidation in the outer segments of cones

Visual Neuroscience ◽

10.1017/s0952523817000013 ◽

2017 ◽

Vol 34 ◽

Cited By ~ 9

Author(s):

SHINYA SATO ◽

RIKARD FREDERIKSEN ◽

M. CARTER CORNWALL ◽

VLADIMIR J. KEFALOV

Keyword(s):

Dark Adaptation ◽

Enzymatic Reaction ◽

Bright Light ◽

Visual Cycle ◽

Cone Photoreceptors ◽

Molecular Identity ◽

Outer Segments ◽

Retinal Chromophore ◽

Enzymatic Machinery ◽

Rod And Cone Photoreceptors

AbstractVertebrate rod and cone photoreceptors require continuous supply of chromophore for regenerating their visual pigments after photoactivation. Cones, which mediate our daytime vision, demand a particularly rapid supply of 11-cis retinal chromophore in order to maintain their function in bright light. An important contribution to this process is thought to be the chromophore precursor 11-cis retinol, which is supplied to cones from Müller cells in the retina and subsequently oxidized to 11-cis retinal as part of the retina visual cycle. However, the molecular identity of the cis retinol oxidase in cones remains unclear. Here, as a first step in characterizing this enzymatic reaction, we sought to determine the subcellular localization of this activity in salamander red cones. We found that the onset of dark adaptation of isolated salamander red cones was substantially faster when exposing directly their outer vs. their inner segment to 9-cis retinol, an analogue of 11-cis retinol. In contrast, this difference was not observed when treating the outer vs. inner segment with 9-cis retinal, a chromophore analogue which can directly support pigment regeneration. These results suggest, surprisingly, that the cis-retinol oxidation occurs in the outer segments of cone photoreceptors. Confirming this notion, pigment regeneration with exogenously added 9-cis retinol was directly observed in the truncated outer segments of cones, but not in rods. We conclude that the enzymatic machinery required for the oxidation of recycled cis retinol as part of the retina visual cycle is present in the outer segments of cones.

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The 9-methyl group of retinal is essential for rapid Meta II decay and phototransduction quenching in red cones

The Journal of General Physiology ◽

10.1085/jgp.200910232 ◽

2009 ◽

Vol 134 (2) ◽

pp. 137-150 ◽

Cited By ~ 14

Author(s):

Maureen E. Estevez ◽

Alexander V. Kolesnikov ◽

Petri Ala-Laurila ◽

Rosalie K. Crouch ◽

Victor I. Govardovskii ◽

...

Keyword(s):

Visual Pigment ◽

Molecular Mechanisms ◽

Bright Light ◽

Rapid Response ◽

Rapid Decay ◽

Rod Photoreceptors ◽

Methyl Group ◽

Response Recovery ◽

Electrophysiological Recordings ◽

Dim Light

Cone photoreceptors of the vertebrate retina terminate their response to light much faster than rod photoreceptors. However, the molecular mechanisms underlying this rapid response termination in cones are poorly understood. The experiments presented here tested two related hypotheses: first, that the rapid decay rate of metarhodopsin (Meta) II in red-sensitive cones depends on interactions between the 9-methyl group of retinal and the opsin part of the pigment molecule, and second, that rapid Meta II decay is critical for rapid recovery from saturation of red-sensitive cones after exposure to bright light. Microspectrophotometric measurements of pigment photolysis, microfluorometric measurements of retinol production, and single-cell electrophysiological recordings of flash responses of salamander cones were performed to test these hypotheses. In all cases, cones were bleached and their visual pigment was regenerated with either 11-cis retinal or with 11-cis 9-demethyl retinal, an analogue of retinal lacking the 9-methyl group. Meta II decay was four to five times slower and subsequent retinol production was three to four times slower in red-sensitive cones lacking the 9-methyl group of retinal. This was accompanied by a significant slowing of the recovery from saturation in cones lacking the 9-methyl group after exposure to bright (>0.1% visual pigment photoactivated) but not dim light. A mathematical model of the turn-off process of phototransduction revealed that the slower recovery of photoresponse can be explained by slower Meta decay of 9-demethyl visual pigment. These results demonstrate that the 9-methyl group of retinal is required for steric chromophore–opsin interactions that favor both the rapid decay of Meta II and the rapid response recovery after exposure to bright light in red-sensitive cones.

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Light-induced changes in GTP and ATP in frog rod photoreceptors. Comparison with recovery of dark current and light sensitivity during dark adaptation.

The Journal of General Physiology ◽

10.1085/jgp.85.1.107 ◽

1985 ◽

Vol 85 (1) ◽

pp. 107-121 ◽

Cited By ~ 60

Author(s):

M S Biernbaum ◽

M D Bownds

Keyword(s):

Dark Current ◽

Dark Adaptation ◽

Recovery Time ◽

Continuous Light ◽

Light Sensitivity ◽

Rod Outer Segments ◽

Rod Photoreceptors ◽

Outer Segments ◽

Atp Levels ◽

Induced Changes

Light decreases GTP and ATP levels in purified suspensions of physiologically active frog rod outer segments still attached to their inner segment ellipsoids (OS-IS). (a) The GTP decrease is slower in OS-IS (t1/2 = 40 s) than in isolated outer segments (t1/2 = 7 s), which suggests there is more effective buffering in OS-IS. (b) The GTP decrease becomes detectable only at intensities greater than those required to saturate the photoresponse. As the intensity of a continuous light is increased over 4 log units, GTP levels decrease linearly with log intensity by as much as 60%. GTP is reduced to steady intermediate levels during extended illumination of intermediate intensity. (c) At levels of illumination bleaching greater than 0.003% of the rhodopsin, a decrease in ATP levels becomes detectable. (d) Following a flash, GTP levels fall and then rise with a recovery time dependent on the intensity of the flash. (e) After both 0.2 and 2% flash bleaches, the recovery of GTP levels parallels the recovery of light sensitivity, which is slower than the recovery of the dark current. This raises the possibility of a link between GTP levels and light sensitivity.

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