In vitro Physiology of Frog Photoreceptor Membranes

Bownds, Deric; Dawes, J.; Miller, Julie Ann

doi:10.1007/978-3-642-85769-0_32

Cited by 12 publications

(6 citation statements)

References 7 publications

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“…lb) Frank et al (4), who showed that there was a roughly linear relationship between the rate of phosphorylation of ROS and the extent of bleaching, but is in contrast to the results of Bownds et al (1), who initially reported that ROS prepared from frog retinas showed maximal phosphorylation after only 1% of the rhodopsin is bleached. In a recent publication, however, they reported that further bleaching did cause an increase in phosphorylation, though small bleaches were much more effective (2,22). Our experiments give no indication of such an effect.…”

Section: Methodscontrasting

confidence: 74%

“…The time course of rhodopsin phosphorylation is much too slow to be involved in the direct response to light but it may be postulated that the phosphorylation is concerned in the adaptation of the photoreceptors to light or dark conditions (5,22). It has been suggested before (25,(27)(28)(29) that the phosphorylation of certain membrane proteins may control passive permeability to certain ions and it may be that the phosphorylation of rhodopsin may similarly control passive permeability in ROS, so mediating the responsiveness to a light impulse.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Light-stimulated phosphorylation of rhodopsin in the retina: the presence of a protein kinase that is specific for photobleached rhodopsin.

Weller¹,

Virmaux²,

Mandel³

1975

Proc. Natl. Acad. Sci. U.S.A.

108

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A protein kinase has been extiacted from bovine rod outer segments by a mild procedure. The enzyme acts specifically on photobleached, not unbleached, rhodopsin and will not catalyze the phosphorylation of histones, phosvitin, or casein. We propose the name "opsin kinase" for the enzyme, which is not affected by cyclic nucleotides but which is inhibited by theophylline. Preparations of purified rod outer segments, however, appear to contain only low concentration of opsin phosphatase activity.It has recently been shown in several laboratories that when rod outer segments (ROS) prepared from frog (1, 2) or ox (3-5) retinas are incubated with ATP and Mg2+ rhodopsin is phosphorylated and the reaction is markedly stimulated by light.There seem to be three ways in which light could act. First, by directly stimulating the activity of the kinase, second, by altering the conformation of the rhodopsin so that it becomes a substrate for the kinase, and third, by altering the concentration of some cofactor which changes the activity of the kinase.In support of the third possibility, it has been observed that the rate of production of 3': 5'-cAMP and cGMP in ROS is lowered on exposure to light (6-10) and, since rates of protein phosphorylation are, in so many cases, controlled by cyclic nucleotides, it was at first thought that the effect of light on cyclic nucleotide concentration could be correlated with the effect on protein phosphorylation (3, 4). A decrease in cyclic nucleotide concentration could increase the net rate of protein phosphorylation if the cyclic nucleotide inhibited a protein kinase or, alternatively, stimulated a protein phosphatase.Cyclic-AMP-inhibited protein kinases have been discovered in the slime mold (11) and cAMP-stimulated protein phosphatases in the toad bladder cell membrane (12).In this paper, we show that neither cAMP nor cGMP have any effect on the phosphorylation of ROS protein. The protein kinase in ROS is specific to photobleached, not unbleached rhodopsin, and the increase in substrate concentration which results from exposure of ROS to light is alone sufficient to account for the increase in protein phosphorylation. MATERIALS AND METHODSPreparation of Rod Outer Segments from Bovine Retinas was as previously described (13). The segments routinely showed a ratio of absorbance at 280 to 500 nm of about 2.2. Incubation with 1 1-cis-retinaldehyde only increased the absorption at 500 nm by 5-10%, indicating that the material was only 5-10% bleached.Abbreviation: ROS, rod outer segments. Reactions were terminated by the addition of 2 ml of ice-cold 10% trichloroacetic acid (or 20% to precipitate histones) and 0.1 mg (0.1 ml) of bovine serum albumin added as protein carrier. The protein precipitate was washed twice with 10% trichloroacetic acid, M orthophosphoric acid, then resuspended in 0.5 ml of 0.1 N NaOH and incubated for 10 min at 37°, after which 2 ml of 10 or 20% trichloroacetic acid was added and the protein was spun down. The protein precipitates were washed once in 1 ml of e...

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Section: Methodscontrasting

confidence: 74%

Section: Discussionmentioning

confidence: 99%

Light-stimulated phosphorylation of rhodopsin in the retina: the presence of a protein kinase that is specific for photobleached rhodopsin.

Weller¹,

Virmaux²,

Mandel³

1975

Proc. Natl. Acad. Sci. U.S.A.

108

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“…Moreover, with this technique, the light-regulated Na + flux is in good quantitative agreement with electrophysiological data. Using different osmotic techniques, Bownds et al (1973Bownds et al ( , 1974 and Brodie and Bownds (1976) found qualitatively comparable results, whereas other investigators have not (Cobbs and Hagins, 1974;Chabre and Cavaggioni, 1975). Therefore, we examined a variety of osmotic techniques.…”

Section: Introductionmentioning

confidence: 84%

Ionic blockage of the light-regulated sodium channels in isolated rod outer segments.

Wormington¹,

Cone²

1978

The Journal of General Physiology

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A B S T R A C T We have investigated, with osmotic techniques, the light-regulated Na + channels in rod outer segments (ROS) and ROS fragments freshly isolated from the frog retina. Values of Na + permeability (PNa) similar to those observed electrophysiologically in the retina were observed using the osmotic technique (continuous flow) described by Korenbrot and Cone. In the other osmotic techniques that we explored, PNa was greatly diminished, if not completely suppressed; however, we found with these techniques that antioxidant conditions (N2 atmosphere or EDTA) significantly increased PNa, suggesting that the Na + channels are highly sensitive to membrane oxidation. Using the continuous flow technique, we investigated the H + and Ca ++ dependence of the Na + channels and found that both of these ions, at micromolar activities, can block the channels. Raising the external H + activity decreases P~a (reversibly) in a single "sigmoidal" response with an apparent pKa of 5.8. Similarly, in the presence of the ionophores X537A or A23187 which allow equilibration of Ca ++ across membranes, the Na + channels are blocked when the external Ca ++ activity is increased from 10 -7 to 10 -s M. This high sensitivity to both H + and Ca ++ ions suggests that high field strength anionic sites may exist in or near the Na + channels and that the channels are blocked when these sites bind H + or Ca ++ ions.

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“…1. (While we have shown previously that washed and purified outer segments show light influenced swelling, the mechanical disruption involved in these preparations does diminish the magnitude of the light effects [5].) The suspension containing approximately l0 s outer segments and their fragments is divided into four to eight 100-/A portions.…”

Section: Methodsmentioning

confidence: 95%

“…Because rhodopsin photochemistry has been extensively studied over the past 30 years (3,4), the photon input to this system can be precisely quantitated. In the past few years several new chemical consequences of illumination have been uncovered: a rhodopsin phosphorylation reaction (5)(6)(7)(8) and changes in the activities of enzymes controlling cyclic nucleotide levels in the cell (9)(10)(11)(12). Photon absorption by rhodopsin ultimately causes sodium channels in the plasma membrane to close; this is generally thought to be effected by the release of an internal transmitter (perhaps calcium) from the discs (13,14).…”

Section: Introductionmentioning

confidence: 99%

Light-sensitive swelling of isolated frog rod outer segments as an in vitro assay for visual transduction and dark adaptation.

Bownds¹,

Brodie²

1975

The Journal of General Physiology

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Frog rod outer segments swell slowly after being shaken from an excised retina into a modified Ringer's solution. The swelling has the following characteristics: (a) It is suppressed by illumination which bleaches only 500 rhodopsin molecules per outer segment per second. This is approximately the level required to saturate the in vivo receptor potential. other inhibitors of mitochondrial electron transport. (g) A process analogous to dark adaptation in vivo can be observed: If 10-50 % of the rhodopsin present is bleached and the outer segments are then kept dark, rapid dark swelling returns after a period of 15-45 min. This swelling is again sensitive to light. We tentatively ascribe the light suppression of swelling to the same decrease in sodium permeability which is observed on illuminating living receptor cells. The experiments suggest that outer segments retain their competence to perform both transduction and dark adaptation after their separation from the retina.

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In vitro Physiology of Frog Photoreceptor Membranes

Cited by 12 publications

References 7 publications

Light-stimulated phosphorylation of rhodopsin in the retina: the presence of a protein kinase that is specific for photobleached rhodopsin.

Light-stimulated phosphorylation of rhodopsin in the retina: the presence of a protein kinase that is specific for photobleached rhodopsin.

Ionic blockage of the light-regulated sodium channels in isolated rod outer segments.

Light-sensitive swelling of isolated frog rod outer segments as an in vitro assay for visual transduction and dark adaptation.

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