Frog rod outer segments with attached inner segment ellipsoids as an in vitro model for photoreceptors on the retina.

Biernbaum, Michael S.; Bownds, M D

doi:10.1085/jgp.85.1.83

Cited by 70 publications

(66 citation statements)

References 44 publications

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“…Retinol fluorescence was excited with 360 nm light using a Xenon continuous arc light source from Sutter Instrument Company (Novato, CA), and the fluorescence image (emission >420 nm) was acquired with a Hamamatsu CCD camera (Hamamatsu Photonics, Hamamatsu-City, Japan). Only metabolically competent cells (23,26) were used for retinol fluorescence imaging experiments. Most of the cells are expected to be the rhodopsin-containing "red" rods, which comprise the overwhelming majority of the rod population in frog retinas (27).…”

Section: Methodsmentioning

confidence: 99%

Interphotoreceptor Retinoid-Binding Protein Is the Physiologically Relevant Carrier That Removes Retinol from Rod Photoreceptor Outer Segments

Blakeley

Cornwall

et al. 2007

Biochemistry

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Light detection by vertebrate rod photoreceptor outer segments results in the destruction of the visual pigment, rhodopsin, as its retinyl moiety is photoisomerized from 11-cis to all-trans. The regeneration of rhodopsin is necessary for vision and begins with the release of the all-trans retinal and its reduction to all-trans retinol. Retinol is then transported out of the rod outer segment for further processing. We used fluorescence imaging to monitor retinol fluorescence and quantify the kinetics of its formation and clearance after rhodopsin bleaching in the outer segments of living isolated frog (Rana pipiens) rod photoreceptors. We independently measured the release of all-trans retinal from bleached rhodopsin in frog rod outer segment membranes and the rate of all-trans retinol removal by the lipophilic carriers interphotoreceptor retinoid binding protein (IRBP) and serum albumin. We find that the kinetics of all-trans retinol formation in frog rod outer segments after rhodopsin bleaching are to a good first approximation determined by the kinetics of all-trans retinal release from the bleached pigment. For the physiological concentrations of carriers, the rate of retinol removal from the outer segment is determined by the IRBP concentration, while the effect of serum albumin is negligible. The results indicate the presence of a specific interaction between IRBP and rod outer segment, probably mediated by a receptor. The effect of different concentrations of IRBP on the rate of retinol removal shows no cooperativity and has an EC 50 of 40 μmol/L.The vertebrate cells responsible for vision are the rod and cone photoreceptors of the retina that convert incoming light to an electrical signal. This conversion takes place in the photoreceptor outer segments, which are full of membrane disks containing the visual pigment, and, in a physiologically important arrangement, are enveloped by the retinal pigment epithelium (RPE). The visual pigment is composed of a chromophore, 11-cis retinal, attached to an integral membrane protein, opsin. The detection of light begins with the absorption of incoming photons by the visual pigment. An absorbed photon isomerizes the chromophore † Supported by NIH/NEI grants EY14850 (YK), EY04939 (RKC) , NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript moiety from 11-cis to all-trans bringing about a conformational change that initiates a cascade of reactions culminating in membrane potential change. The recovery of the cell from light involves the deactivation of the intermediates activated by light, and the reestablishment of membrane potential (1,2). However, the isomerized chromophore, all-trans retinal, remains. For vision to be possible, it is essential that the visual pigment regenerate: that is, the alltrans retinal has to be removed, and fresh 11-cis retinal has to be provided to combine with opsin and reform the visual pigment. The reactions regenerating the pigment are known as the Visual Cycle (3-5).In the case of the rod photoreceptors, t...

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Section: Methodsmentioning

confidence: 99%

Interphotoreceptor Retinoid-Binding Protein Is the Physiologically Relevant Carrier That Removes Retinol from Rod Photoreceptor Outer Segments

Blakeley

Cornwall

et al. 2007

Biochemistry

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“…Afterwards, retinas were transferred to 3 ml of fresh standard Ringer's solution. They were then gently swirled and the surface of the scleral side gently puckered as described by Biernbaum and Bownds (1985). This movement effectively released solitary photoreceptors or small patches of these cells.…”

Section: Cell Preparationmentioning

confidence: 99%

Lactate released by Muller glial cells is metabolized by photoreceptors from mammalian retina

1995

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The nature of fuel molecules trafficking between mammalian glial cells and neurons was explored using acute retinal cell preparations of solitary Muller glial cells, Muller cells still attached to photoreceptors (the “cell complex”), and solitary photoreceptors. 14C- Molecules in the cell complex, Muller cells, and respective baths were quantitated following 30 min incubation in bicarbonate-buffered Ringer's solution carrying 5 mM 14C(U)-glucose, and substrate preference by solitary photoreceptors was assessed by measuring 14CO2 production. Muller cells alone metabolized 14C-glucose predominantly to carbohydrate intermediates, while the presence of photoreceptors raised proportionately the amount of radiolabeling in amino acids. 14C-Lactate was the major carbohydrate found in the bath. However, in the presence of photoreceptors, its amount was 70% less than that for Muller cells alone. This decrease matched the expected production of 14CO2 by photoreceptor oxidative metabolism and was antagonized by the addition of unlabeled lactate. Moreover, while solitary photoreceptors consumed both exogenous 14C-lactate and 14C-glucose, lactate was a better substrate for their oxidative metabolism. In the cell complex, the metabolism of amino acids increased and illumination affected primarily glutamate and glutamine production: the specific activity of glutamate changed in parallel with that of lactate, and that of glutamine increased by eightfold in darkness. These results demonstrate transfer of lactate from Muller cells to photoreceptors and underscore a photoreceptor-dependent modulation of lactate and amino acid metabolism. We propose that net production and release of lactate by Muller cells serves to maintain their glycolysis elevated and to fuel mitochondrial oxidative metabolism and glutamate resynthesis in photoreceptors.

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“…These values for the ATP and GTP content are comparable with those obtained from frozen sections of rabbit and monkey retinas (Berger, DeVries, Carter, Schulz, Passonneau, Lowry & Ferendelli, 1980), and with those obtained from frog r.o.s. preparations that contain part of the inner segment and show normal photocurrents (Biernbaum & Bownds, 1985). The above values are over-all concentrations based on a rhodopsin concentration of 3 mm in r.o.s.…”

Section: Metabolic Requirements Of Ca2+ Fluxesmentioning

confidence: 99%

“…The large majority of this Ca2+ is bound to Ca2+ buffer sites inside disks in equilibrium with free internal Ca2+ concentrations in the micromolar range (Schnetkamp, 1979;Schnetkamp & Kaupp, 1985). Disk membranes contain at least two pathways to account for rapid transport of Ca2+ across the disk membrane: Na-Ca exchange (Schnetkamp, Daemen & Bonting, 1977;Biernbaum & Bownds, 1985; Fig. 9), and a cyclic-GMP-dependent pathway (Caretta & Cavaggioni, 1983;Kaupp & Koch, 1984;Fig.…”

Section: Metabolic Requirements Of Ca2+ Fluxesmentioning

confidence: 99%

Sodium‐calcium exchange in the outer segments of bovine rod photoreceptors.

Schnetkamp¹

1986

The Journal of Physiology

100

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SUMMARY1. Intact rod outer segments (r.o.s.) isolated from bovine retinas were used to measure net Ca2+ fluxes using the optical Ca2+ indicator Arsenazo III. Ca2+ fluxes were observed, which could change the internal Ca2+ content of isolated r.o.s. by as much as 0 5 mM s-5.2. The Ca2+ content of isolated intact r.o.s. was strongly dependent on the Na/Ca ratio in the isolation medium, and could be made less than 01 mol Ca2+ mold1 rhodopsin (zero Ca2+ in isolation medium) or up to 7 mol Ca2+ mold1 rhodopsin (zero Na+ in isolation medium).3. Ca2+ efflux from r.o.s. rich in Ca2+ was observed only when Na+ was added to the external medium (as opposed to any other alkali cation); in Ca2+-depleted r.o.s. Ca2+ uptake required the presence of internal Na+ and was inhibited selectively by external Na+. These results suggest that Na-Ca exchange across the plasma membrane operated freely in both directions and controlled the internal Ca2+ concentration in r.o.s.4. Na+-stimulated Ca2+ efflux depended on the external Na+ concentration in a sigmoidal way. This suggests that the simultaneous binding of two Na ions is rate limiting for transport.5. In Ca2+-depleted r.o.s. and in the absence of external Na+, 1 mol Ca2+ mold1 rhodopsin (or 3 mM-total Ca2+) could be taken up within 1 min by intact r.o.s. at a free external Ca2+ concentration of about 1 /tM. 6. Only part of the internal Ca2+ was available for Na-Ca exchange. The external Na+ and K+ concentration as well as the temperature were factors controlling the accessibility of internal Ca2+ to participate in Na-Ca exchange.7. Ca2+ fluxes in r.o.s. with a permeabilized plasma membrane but intact disk membranes were very similar to those observed in intact r.o.s.; Na-Ca exchange could operate in both directions across the disk membrane.8. In addition to Na-Ca exchange, leaky r.o.s. also showed a guanosine 3', 5'-cyclic monophosphate (cyclic GMP)-induced Ca2+ release that was about I of the rate of Present address:

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Frog rod outer segments with attached inner segment ellipsoids as an in vitro model for photoreceptors on the retina.

Cited by 70 publications

References 44 publications

Interphotoreceptor Retinoid-Binding Protein Is the Physiologically Relevant Carrier That Removes Retinol from Rod Photoreceptor Outer Segments

Interphotoreceptor Retinoid-Binding Protein Is the Physiologically Relevant Carrier That Removes Retinol from Rod Photoreceptor Outer Segments

Lactate released by Muller glial cells is metabolized by photoreceptors from mammalian retina

Sodium‐calcium exchange in the outer segments of bovine rod photoreceptors.

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