scholarly journals Light-dependent binding of G-protein to outer segment membranes of toad photoreceptors.

1986 ◽  
Vol 88 (5) ◽  
pp. 675-694 ◽  
Author(s):  
N J Mangini ◽  
D R Pepperberg ◽  
W Baehr
Keyword(s):  
G Protein ◽  
Visual Pigment ◽  
Outer Segment ◽  
Tight Binding ◽  
Beta Subunits ◽  
Hypotonic Medium ◽  
Intense Light ◽  
Low Levels ◽  
Toad Bufo ◽  
Peak Value

Light-dependent changes in the binding of G-protein were analyzed in outer segment disk membranes obtained from photoreceptors of the toad (Bufo marinus) retina. Isolated, intact retinas, incubated in oxygenated Ringer's solution at 23 +/- 1 degree C, were subjected to various conditions of illumination and then incubated in darkness for specified periods. The retinas were then chilled (0-4 degrees C) and the receptor outer segments (ROS) were isolated. Binding of the alpha- and beta-subunits of G-protein to the ROS membranes was analyzed by quantitating G alpha and G beta extracted from the membranes with hypotonic medium lacking GTP vs. hypotonic medium containing GTP (H and HG extracts, respectively). For retinas illuminated and then immediately chilled for analysis, the extent of G binding (relative abundance of G alpha, beta in the HG extract) increased with the extent of bleaching of the visual pigment. Near-maximal binding was observed after bleaches of greater than or equal to 30%. With an increasing period of incubation in darkness after approximately 70% bleaching, the extent of binding declined gradually to low levels characteristic of unbleached retinas. The period required for half-completion of the decline was approximately 10(3) s. A gradual decline in G binding, from a rapidly developing peak value, was also observed with an increasing period of exposure to intense light. Viewed in the context of previous electrophysiological data, our results indicate that sustained bleaching desensitization of the rods does not depend upon a persisting state of "tight binding" (immobilization) of G-protein by bleached visual pigment.

Dexamethasone increases G alpha q-11 expression and hormone-stimulated phospholipase C activity in UMR-106-01 cells.

1997 ◽  
Vol 273 (3) ◽  
pp. E528 ◽  
Author(s):  
J Mitchell ◽  
A Bansal
Keyword(s):  
Adenylyl Cyclase ◽  
Phospholipase C ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Beta Subunits ◽  
Adenylyl Cyclase Activity ◽  
Protein Subunits ◽  
G Alpha Q ◽  
Umr 106 ◽  
G Protein Coupled

Glucocorticoids regulate responsiveness of many cells to hormones that bind to G protein-coupled receptors. We examined the effect of glucocorticoids on parathyroid hormone (PTH) activation of two G protein-activated signal transduction pathways, phospholipase C (PLC) and adenylyl cyclase, in osteosarcoma UMR-106-01 cells. Dexamethasone (100 nM) increased PTH-stimulated and NaF-stimulated PLC activity by > 100% over 4 days (223 +/- 8 and 293 +/- 8.2% of control after 4 days for PTH and NaF-stimulated activity, respectively). The increase in PTH-stimulated adenylyl cyclase response in the same cells was more modest (162 +/- 5.4 and 171 +/- 6.8% of control after 4 days for PTH and NaF-stimulated activity, respectively). PTH activation of PLC was blocked by antiserums to G alpha q-11 and activation of adenylyl cyclase by G alpha s antiserums. Quantification of these G protein subunits in control and dexamethasone-treated cells showed a 78% increase in G alpha q-11 (from 18.1 +/- 1.2 to 32.2 +/- 1.5 pmol/mg), whereas G alpha s was increased only 34% (from 6.2 +/- 0.5 to 8.2 +/- 0.3 pmol/mg) and G beta-subunits were increased 40% (from 54 +/- 2.3 to 75.2 +/- 3.8 pmol/mg). These results suggest that glucocorticoids are more potent regulators of PLC activity than adenylyl cyclase activity in UMR cells, and this is mediated, at least in part, by differential increases in G alpha q-11 proteins.

The binding of G-protein to rod outer segment phospholipids at the nitrogen–water interface

1989 ◽  
Vol 67 (8) ◽  
pp. 422-427 ◽  
Author(s):  
C. Salesse ◽  
F. Lamarche ◽  
R. M. Leblanc
Keyword(s):  
G Protein ◽  
Aqueous Phase ◽  
Surface Pressure ◽  
Outer Segment ◽  
Membrane Surface ◽  
Water Interface ◽  
Rod Outer Segment ◽  
Visual Process ◽  
Adsorption Desorption ◽  
Hopping Model

In the visual process, one photoexcited rhodopsin (R*) catalyzes the activation of hundreds of G-proteins. It remains to be determined whether G-protein and R* find one another by membrane surface diffusion of these components (diffusion model) or by diffusion of G-protein through the aqueous phase (hopping model). A monolayer of each main rod outer segment (ROS) phospholipid interacting with a subphase containing G-protein, has been used to simulate the interaction of G-protein with the cytoplasmic surface of discal membranes. The possible diffusion of G-protein through the aqueous phase was then measured by observing its adsorption–desorption in the monolayer of each main ROS phospholipid. From examination of surface pressure and ellipsometric isotherms at the nitrogen–water interface, we have determined that once incorporated into the monolayer, the G-protein remains associated, independent of surface pressure, thus providing evidence against the hopping model.Key words: rod outer segment, visual process, hopping model, G-protein, phospholipid monolayer, ellipsometry.

Extraction and characterization of the blue-sensitive visual pigment of the toad Bufo marinus

1987 ◽  
Vol 65 (4) ◽  
pp. 884-887 ◽  
Author(s):  
A. J. Sillman
Keyword(s):  
Vitamin A ◽  
Visual Pigment ◽  
Chemical Properties ◽  
Bufo Marinus ◽  
Physical And Chemical Properties ◽  
Electrophysiological Studies ◽  
Physical And Chemical ◽  
Toad Bufo ◽  
And Chemical Properties

The blue-sensitive visual pigment of the green rods of Bufo marinus was extracted with digitonin. The pigment is present in an amount equal to about 11% that of rhodopsin. It is based on vitamin A1 and exhibits a maximum absorbance of 433 nm. The pigment is labile and readily destroyed by hydroxylamine, regenerates to a much greater degree than does rhodopsin, and is more effectively extracted from the membrane than is rhodopsin. The green rod pigment of Bufo marinus appears to share the same physical and chemical properties as the green rod pigments of other amphibians. Therefore, the results of electrophysiological studies on the green rods of Bufo marinus can be more confidently generalized to other species. The results are discussed in terms of the blue phototaxis that is characteristic of many amphibians.

Effect of hydroxylamine on the subcellular distribution of arrestin (S-antigen) in rod photoreceptors

1994 ◽  
Vol 11 (3) ◽  
pp. 561-568 ◽  
Author(s):  
Nancy J. Mangini ◽  
Grady L. Garner ◽  
Tinging L. Okajima ◽  
Larry A. Donoso ◽  
David R. Pepperberg
Keyword(s):  
Visual Pigment ◽  
Driving Force ◽  
Outer Segment ◽  
Processing System ◽  
Bright Light ◽  
Photoreceptor Outer Segment ◽  
Rod Photoreceptors ◽  
Average Value ◽  
Secondary Antibodies ◽  
Microscopic Image Processing

AbstractThe immunocytochemical labeling of arrestin (S-antigen) in photoreceptors of the ovine retina was examined following incubation of the retina with hydroxylamine (NH2OH), an agent known to inhibit the phosphorylation of photoactivated rhodopsin. Intact, isolated retinas bathed in medium containing 20 mM NH2OH, or in control medium lacking NH2OH, were maintained in darkness or exposed to bright light for 3 min (dark-adapted and light-adapted conditions, respectively); further incubated in darkness for 10 min; and then fixed and prepared for cryosectioning. Cryosections were incubated with anti-S-antigen monoclonal antibody MAb A2G5; with secondary antibodies that were conjugated with horseradish peroxidase; and with either 3–amino-9–ethyl carbazole or diaminobenzidine as chromogen. Anti-arrestin labeling in cryosections was then analyzed densitometrically using a light-microscopic image processing system. In dark-adapted control retinas, labeling density of the photoreceptor outer segment (OS) layer (0.061 ± 0.004; average ± S.e.m.) was less than that of the inner segment (IS) layer (0.138 ± 0.011). In light-adapted control retinas, OS labeling density (0.139 ± 0.007) exceeded IS labeling density (0.095 ± 0.005). Incubation with NH2OH eliminated this light-dependent increase in labeling of the OS relative to that of the IS, i.e. eliminated the increase in relative OS/IS labeling. Densities of labeling were 0.110 ± 0.006 (OS) and 0.183 ± 0.006 (IS) in NH2OH-treated dark-adapted retinas vs. 0.078 ± 0.004 (OS) and 0.182 ± 0.008 (IS) in NH2OH-treated light-adapted retinas. Anti-arrestin labeling was also examined in retinas that were exposed to 3 min or 13 min of bright light and then immediately fixed. Among retinas incubated in the absence of NH2OH, an increase in OS/IS labeling density was evident after 3 min of illumination, and retinas illuminated for 13 min exhibited an even larger increase in OS/IS labeling. An increase in OS/IS labeling was also exhibited by NH2OH-treated retinas that had been illuminated for 3 min; by comparison with dark-adapted NH2OH-treated controls (average value of OS/IS labeling: 0.60), OS/IS labeling in these illuminated retinas was 0.97. However, OS/IS labeling in NH2OH-treated retinas that had been illuminated for 13 min (average value: 0.35) was lower than that of the dark-adapted controls. The results indicate that, within intact rods, NH2OH inhibits the light-dependent increase in OS/IS anti-arrestin labeling that is ordinarily expressed at long times (~10 min) after major bleaching of the visual pigment. Among the possible bases for the effect of NH2OH are a reduction in the driving force for the movement of arrestin from the inner to the outer segment and/or a facilitation of the degradation of arrestin in the outer segment.

scholarly journals Utilization of retinoids in the bullfrog retina.

1982 ◽  
Vol 80 (6) ◽  
pp. 885-913 ◽  
Author(s):  
J I Perlman ◽  
B R Nodes ◽  
D R Pepperberg
Keyword(s):  
Vitamin A ◽  
Visual Pigment ◽  
Outer Segment ◽  
Rana Catesbeiana ◽  
Retinyl Palmitate ◽  
Visual Cycle ◽  
Trans Isomers ◽  
Isolated Retina ◽  
Cis Isomer

The capacity to generate 11-cis retinal from retinoids arising naturally in the eye was examined in the retina of the bullfrog, Rana catesbeiana. Retinoids, co-suspended with phosphatidylcholine, were applied topically to the photoreceptor surface of the isolated retina after substantial bleaching of the native visual pigment. The increase in photoreceptor sensitivity associated with the formation of rhodopsin, used as an assay for the appearance of 11-cis retinal in the receptors, was analyzed by extracellular measurement of the photoreceptor potential; in separate experiments using the isolated retina or receptor outer segment preparations, the formation of rhodopsin was measured spectrophotometrically. Treatments with the 11-cis isomers of retinal and retinol induced significant increases in both the rhodopsin content and photic sensitivity of previously bleached receptors. The all-trans isomers of retinyl palmitate, retinol, and retinal, as well as the 11-cis isomer of retinyl palmitate, were inactive by both the electrophysiological and spectrophotometric criteria for the generation of rhodopsin. Treatment with any one of the "inactive" retinoids did not abolish the capacity of subsequently applied 11-cis retinal or 11-cis retinol to promote the formation of rhodopsin. The data are discussed in relation to the interconversions of retinoids ("visual cycle of vitamin A") thought to mediate the regeneration of rhodopsin in vivo after extensive bleaching.

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