Axial diffusion of retinol in isolated frog rod outer segments following substantial bleaches of visual pigment

doi:10.1016/0042-6989(89)90132-6

Vision Research

Volume 29, Issue 11, 1989, Pages 1485-1492

https://doi.org/10.1016/0042-6989(89)90132-6 Get rights and content

Abstract

Partial bleaches of rhodopsin were made in either the proximal or distal halves of isolated Rana pipiens rod outer segments. The fluorescence of all-trans retinol was recorded 15, 60 and 120 min following 17% and 63% bleaches. Some of the retinol that formed remained immobilized in the bleached halves of the outer segments, while another portion was slowly mobilized and diffused along the cell axis.

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Cited by (11)

All-trans retinol in rod photoreceptor outer segments moves unrestrictedly by passive diffusion
2006, Biophysical Journal
Citation Excerpt :
Previous studies (17,18) took advantage of the fluorescence of all-trans retinol to monitor its movement, whereas others (19) were based on its distinctive light absorption properties. Because of the technical limitations at the time, these studies were largely qualitative, but they did seem to indicate that the all-trans retinol produced locally after rhodopsin bleaching can move axially within the rod outer segment (18,19). We have used two-photon excitation (720 nm) of retinol fluorescence to monitor the concentration of retinol in frog rod outer segments.
The visual pigment protein of vertebrate rod photoreceptors, rhodopsin, contains an 11-cis retinyl moiety that is isomerized to all-trans upon light absorption. Subsequently, all-trans retinal is released from the protein and reduced to all-trans retinol, the first step in the recycling of rhodopsin’s chromophore group through the series of reactions that constitute the visual cycle. The concentration of all-trans retinol in photoreceptor outer segments can be monitored from its fluorescence. We have used two-photon excitation (720 nm) of retinol fluorescence and fluorescence recovery after photobleaching to characterize the mobility of all-trans retinol in frog photoreceptor outer segments. Retinol produced after rhodopsin bleaching moved laterally in the disk membrane bilayer with an apparent diffusion coefficient of 2.5 ± 0.3 μm² s⁻¹. The diffusion coefficient of exogenously added retinol was 3.2 ± 0.5 μm² s⁻¹. These diffusion coefficients are in close agreement with those reported for lipids, suggesting that retinol is not tightly bound to protein sites that would be diffusing much more slowly in the plane of the membrane. In agreement with this interpretation, a fluorescent-labeled C-16 fatty acid diffused laterally with a similar diffusion coefficient, 2.2 ± 0.2 μm² s⁻¹. Retinol also moved along the length of the rod outer segment, with an apparent diffusion coefficient of 0.07 ± 0.01 μm² s⁻¹, again suggesting that it is not tightly bound to proteins that would confine it to the disks. The axial diffusion coefficient of exogenously added retinol was 0.05 ± 0.01 μm² s⁻¹. In agreement with passive diffusion, the rate of axial movement was inversely proportional to the square of the length of the rod outer segment. Diffusion of retinol on the plasma membrane of the outer segment can readily account for the measured value of the axial diffusion coefficient, as the plasma membrane comprises ∼1% of the total outer-segment membrane. The values of both the lateral and axial diffusion coefficients are consistent with most of the all-trans retinol in the outer segments moving unrestricted and not being bound to carrier proteins. Therefore, and in contrast to other steps of the visual cycle, there does not appear to be any specialized processing for all-trans retinol within the rod outer segment.
Retinoid cycle in the vertebrate retina: Experimental approaches and mechanisms of isomerization
2003, Vision Research
Retinoid cycle describes a set of chemical transformations that occur in the photoreceptor and retinal pigment epithelial cells. The hydrophobic and labile nature of the retinoid substrates and the two-cell chromophore utilization-regeneration system imposes significant constraints on the experimental biochemical approaches employed to understand this process. A brief description of the recent developments in the investigation of the retinoid cycle is the current topic, which includes a review of novel results and techniques pertaining to the retinoid cycle. The chemistry of the all-trans-retinol to 11-cis-retinol isomerization is also discussed.
Confronting complexity: The interlink of phototransduction and retinoid metabolism in the vertebrate retina
2001, Progress in Retinal and Eye Research
Absorption of light by rhodopsin or cone pigments in photoreceptors triggers photoisomerization of their universal chromophore, 11-cis-retinal, to all-trans-retinal. This photoreaction is the initial step in phototransduction that ultimately leads to the sensation of vision. Currently, a great deal of effort is directed toward elucidating mechanisms that return photoreceptors to the dark-adapted state, and processes that restore rhodopsin and counterbalance the bleaching of rhodopsin. Most notably, enzymatic isomerization of all-trans-retinal to 11-cis-retinal, called the visual cycle (or more properly the retinoid cycle), is required for regeneration of these visual pigments. Regeneration begins in rods and cones when all-trans-retinal is reduced to all-trans-retinol. The process continues in adjacent retinal pigment epithelial cells (RPE), where a complex set of reactions converts all-trans-retinol to 11-cis-retinal. Although remarkable progress has been made over the past decade in understanding the phototransduction cascade, our understanding of the retinoid cycle remains rudimentary. The aim of this review is to summarize recent developments in our current understanding of the retinoid cycle at the molecular level, and to examine the relevance of these reactions to phototransduction.
Retinol kinetics in the isolated retina determined by retinoid extraction and HPLC
1997, Experimental Eye Research
Suzuki et al. [Vis. Res.26, 425–9 (1986);Vis. Res.28, 1061–70 (1988)] have described a formaldehyde-based (HCHO-based) extraction procedure that efficiently recovers 11-cisretinal initially present as rhodopsin chromophore in photoreceptor membranes. Using the isolated retina of the toad (Bufo marinus), we tested whether this procedure (‘HCHO’ method), in combination with a formaldehyde-free extraction procedure (‘i/h’ method) and the analysis of extracted retinoids by high performance liquid chromatography (HPLC), can account quantitatively for light-induced changes in retinoid levels and thus serve as an alternative to spectrophotometry for tracking the formation of all-transretinol in this intact rod preparation. Initially dark-adapted retinas were incubated in bright light or in darkness and then analysed by homogenization and extraction using the HCHO and i/h methods. Combined data obtained using the two extraction procedures indicated a near-conservation of total retinoid recovered from dark-incubated and illuminated retinas, and thus accounted for light-induced changes in retinoid levels. The HCHO procedure, employing formaldehyde, isopropanol and hexane, was similar to that described by Suzuki et al. and recovered retinaldehydes including chromophoric 11-cisretinal. The i/h procedure utilized isopropanol and hexane and, unlike the HCHO method, efficiently recovered all-transretinol. Illumination (onset at time zero) that produced an approximately exponential decline of 11-cisretinal (time constant of 24 s) led to an increase and then a gradual decline in all-transretinal. The normalized peak level of all-transretinal, representing about 0.54 of the total molar quantity of recovered retinoid, developed with illumination periods of 10–80 s. The normalized level of all-transretinol reached ≃0.3 in retinas illuminated for 1 min and, with longer illuminations (up to 30 min), exhibited an approximately exponential further growth to ≃0.9 with a time constant of 9.2 min. The results indicate the workability of the HCHO and i/h extraction procedures for tracking the in situ conversion of all-transretinal to all-transretinol, a reaction thought to be important for both operation of the retinoid visual cycle and shut-off of the phototransduction cascade.
Multiphoton imaging of the retina
2017, Handbook of Visual Optics: Instrumentation and Vision Correction: Volume II
Images of photoreceptors in living primate eyes using adaptive optics two-photon ophthalmoscopy
2011, Biomedical Optics Express

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Axial diffusion of retinol in isolated frog rod outer segments following substantial bleaches of visual pigment

Abstract

Biochimica et Biophysica Acta

Journal of Biological Chemistry

Experimental Eye Research

Vision Research

Vision Research

Journal of Biological Chemistry

Journal of Biological Chemistry

Biophysical Journal

Biophysical Chemistry

Biophysical Journal

Archives of Biochemistry and Biophysics

Biochemical and Biophysical Research Communications

Journal of Biological Chemistry

Biochemical and Biophysical Research Communications

Vision Research

Regeneration of rhodopsin in frog rod outer segments

Journal of Physiology, London