Hydroxylamine-dependent inhibition of rhodopsin phosphorylation in the isolated retina

doi:10.1016/0014-4835(92)90049-X

Experimental Eye Research

Volume 54, Issue 3, March 1992, Pages 369-376

https://doi.org/10.1016/0014-4835(92)90049-X Get rights and content

Abstract

Hydroxylamine (NH₂OH), a substance known to accelerate the decay of the metarhodopsin II bleaching intermediate of rhodopsin, was examined for its effect on the light-dependent phosphorylation of rhodopsin in the intact, isolated retina. Groups of ovine and bovine retinas that had been pre-incubated in darkness with ³²P-inorganic phosphate were supplemented with NH₂OH at final concentrations of up to 20 mm, then irradiated and further incubated in darkness. Rod outer segments isolated from the incubated retinas were subjected to SDS-PAGE; the gel was analysed for ³²P (autoradiography) and protein (Coomassie staining), to determine the specific radioactivity (ratio of ³²P and protein levels; ‘³²P/opsin’) of the opsin monomer band. Among retinas of a given experimental group, ³²P/opsin declined with increasing concentration of added NH₂OH. The relative value of ³²P/opsin exhibited by controls (0 mm NH₂OH) was halved in the presence of about 1–2 mm NH₂OH, and was reduced by ≥ 80% in the presence of 20 mm NH₂OH. Supplementation of the retina with 20 mm NH₂OH 1 min after irradiation caused relatively little reduction in ³²P/opsin. The results indicate that the light-dependent phosphorylation of rhodopsin in situ is substantially inhibited by NH₂OH at millimolar levels. The data are discussed in relation to previous electrophysiological studies that have examined rod dark adaptation in NH₂OH-treated retinas.

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Melanopsin is highly resistant to light and chemical bleaching in vivo
2012, Journal of Biological Chemistry
Citation Excerpt :
Although 60 mm hydroxylamine treatment led to a consistently moderate to large loss of light sensitivity in ipRGCs and 30 mm hydroxylamine showed inconsistently sized losses, we believe these changes were mostly due to off-target effects as they coincided with decreased RGC firing in response to high K+ concentrations. Above 50 mm, hydroxylamine has been shown to cause significant disruptions in G-protein-coupled receptor phosphorylation and Gqα palmitoylation-mediated membrane localization (44, 45). These and potentially other nonspecific effects of hydroxylamine could lead to the apparent loss of light sensitivity seen here as well as the inability of all ganglion cells to fire in response to an increased KCl concentration.
Melanopsin is the photopigment of mammalian intrinsically photosensitive retinal ganglion cells, where it contributes to light entrainment of circadian rhythms, and to the pupillary light response. Previous work has shown that the melanopsin photocycle is independent of that used by rhodopsin (Tu, D. C., Owens, L. A., Anderson, L., Golczak, M., Doyle, S. E., McCall, M., Menaker, M., Palczewski, K., and Van Gelder, R. N. (2006) Inner retinal photoreception independent of the visual retinoid cycle. Proc. Natl. Acad. Sci. U.S.A. 103, 10426–10431). Here we determined the ability of apo-melanopsin, formed by ex vivo UV light bleaching, to use selected chromophores. We found that 9-cis-retinal, but not all-trans-retinal or 9-cis-retinol, is able to restore light-dependent ipRGC activity after bleaching. Melanopsin was highly resistant to both visible-spectrum photic bleaching and chemical bleaching with hydroxylamine under conditions that fully bleach rod and cone photoreceptor cells. These results suggest that the melanopsin photocycle can function independently of both rod and cone photocycles, and that apo-melanopsin has a strong preference for binding cis-retinal to generate functional pigment. The data support a model in which retinal is continuously covalently bound to melanopsin and may function through a reversible, bistable mechanism.
Enhanced shutoff of phototransduction in transgenic mice expressing palmitoylation-deficient rhodopsin
2005, Journal of Biological Chemistry
Palmitoylation is a reversible, post-translational modification observed in a number of G-protein-coupled receptors. To gain a better understanding of its role in visual transduction, we produced transgenic knock-in mice that expressed a palmitoylation-deficient rhodopsin (Palm^–/–). The mutant rhodopsin was expressed at wild-type levels and showed normal cellular localization to rod outer segments, indicating that neither rhodopsin stability nor its intracellular trafficking were compromised. But Palm^–/– rods had briefer flash responses and reduced sensitivity to flashes and to steps of light. Upon exposure to light, rhodopsin became phosphorylated at a faster rate in mutant than in wild-type retinas. Since quench of rhodopsin begins with its phosphorylation, these results suggest that palmitoylation may modulate rod photoreceptor sensitivity by permitting rhodopsin to remain active for a longer period.
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.
Hydroxylamine blocks adenosine A<inf>1</inf> receptor-mediated inhibition of synaptic transmission in rat hippocampus
1999, Brain Research
The nitric oxide donor hydroxylamine (NH₂OH) induced a transient depression of the evoked synaptic potential recorded in the rat hippocampal CA1 region. This depression was abolished with an adenosine A₁ antagonist, 8-cyclopentyltheophylline. In addition, hydroxylamine reversed adenosine A₁ receptor-mediated inhibition of the evoked population spike, the fEPSP and the intracellularly recorded EPSP. The inhibitory modulation of adenosine A₁ receptor activation by hydroxylamine suggests the presence of a potent endogenous regulatory site.
Phosphorylation of non-bleached rhodopsin in intact retinas and living frogs
1996, Journal of Biological Chemistry
The photoresponse in retinal photoreceptors begins when a molecule of rhodopsin is excited by a photon of light. Photoexcited rhodopsin activates an enzymatic cascade including the G-protein transducin and cyclic GMP phosphodiesterase. As a result, cytoplasmic cyclic GMP concentration is decreased and the photoresponse is initiated. This process is terminated when rhodopsin is phosphorylated by rhodopsin kinase and subsequently blocked by a protein called arrestin. It has been noted by several investigators that light can cause phosphorylation of not only photoexcited but also non-excited rhodopsin in rod photoreceptors. A goal of this study was to determine how much non-bleached rhodopsin is phosphorylated. To determine how the structural integrity of the photoreceptor influences the extent of non-bleached rhodopsin phosphorylation, we studied the reaction in electropermeabilized rod outer segments, in rod outer segments still attached to isolated retinas and in living frogs. In the first two preparations, we found that the maximum extent of non-bleached rhodopsin phosphorylation was approximately 1% of the total rhodopsin pool. In living frogs, the maximal amount of non-bleached rhodopsin phosphorylation was ∼2% of the total rhodopsin pool and occurred after prolonged illumination by the relatively dim light intensity of 20 lux. These data appear to exclude models for light adaptation that postulate high levels of phosphorylation of non-bleached rhodopsin in rod photoreceptors.
Depalmitoylation of rhodopsin with hydroxylamine
1995, Methods in Enzymology
This chapter describes the procedures for the in vivo labeling of rhodopsin with [³H]palmitate and the removal of palmitate from rhodopsin in vitro using hydroxylamine (NH₂OH). The properties of hydroxylamine-depalmitoylated rhodopsin exhibited in assays of regeneration and transducin activation, as well as information obtained by others using the complementary approach of site-directed mutagenesis to eliminate the palmitate groups from opsin is summarized in the chapter. Rhodopsin plays a critical role in visual transduction, the process that links the absorption of light with generation of an electrical response within the rod. Proteins containing palmitate include p21^ras, viral glycoproteins, and mammalian transferrin receptor. Palmitoylation occurs posttranslationally, either in the Golgi apparatus or in the endoplasmic reticulum. The palmitoylation is a cotranslational or early posttranslational modification of rat rhodopsin. However, the nonenzymatic exchange of palmitate can be demonstrated in isolated rod outer segment (ROS¹³).

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Hydroxylamine-dependent inhibition of rhodopsin phosphorylation in the isolated retina

Abstract

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

Vision Res.

Vision Res.

Biochim. Biophys. Acta

FEBS Lett.

FEBS Lett.

Vision Res.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Neurochemistry (Great Britain)

Exp. Eye Res.

FEBS Lett.

J. Biol. Chem.

Illumination of bovine photoreceptor membranes causes phosphorylation of both bleached and unbleached rhodopsin molecules

Biochemistry

Isolation and identification of the phosphorylated species of rhodopsin

Biochemistry

Inactivation of photoexcited rhodopsin in retinal rods: the roles of rhodopsin kinase and 48-kDa protein (arrestin)

Biochemistry

The transitory complex between photoexcited rhodopsin and transducin. Reciprocal interaction between the retinal site in rhodopsin and the nucleotide site in transducin

Eur. J. Biochem.

Phosphorylation of frog photoreceptor membranes induced by light

Nature New Biol.

Rhodopsin photoproducts and rod sensitivity in the skate retina

J. Gen. Physiol.

Light induced shift and binding of S-antigen in retinal rods

Curr. Eye Res.

The links between rhodopsin bleaching and visual adaptation

Biochem. Soc. Trans.

Molecular mechanism of visual transduction

Eur. J. Biochem.