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Onset of feedback reactions underlying vertebrate rod photoreceptor light adaptation.

Calvert PD, Ho TW, LeFebvre YM, Arshavsky VY - J. Gen. Physiol. (1998)

Bottom Line: Light adaptation in vertebrate photoreceptors is thought to be mediated through a number of biochemical feedback reactions that reduce the sensitivity of the photoreceptor and accelerate the kinetics of the photoresponse.Guanylate cyclase activity and rhodopsin phosphorylation respond to changes in Ca2+ very rapidly, on a subsecond time scale.Therefore, cGMP-dependent regulation of transducin GTPase is likely to occur only during prolonged bright illumination.

View Article: PubMed Central - PubMed

Affiliation: Howe Laboratory of Ophthalmology, Harvard Medical School and the Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114, USA. pdcalvert@meei.harvard.edu

ABSTRACT
Light adaptation in vertebrate photoreceptors is thought to be mediated through a number of biochemical feedback reactions that reduce the sensitivity of the photoreceptor and accelerate the kinetics of the photoresponse. Ca2+ plays a major role in this process by regulating several components of the phototransduction cascade. Guanylate cyclase and rhodopsin kinase are suggested to be the major sites regulated by Ca2+. Recently, it was proposed that cGMP may be another messenger of light adaptation since it is able to regulate the rate of transducin GTPase and thus the lifetime of activated cGMP phosphodiesterase. Here we report measurements of the rates at which the changes in Ca2+ and cGMP are followed by the changes in the rates of corresponding enzymatic reactions in frog rod outer segments. Our data indicate that there is a temporal hierarchy among reactions that underlie light adaptation. Guanylate cyclase activity and rhodopsin phosphorylation respond to changes in Ca2+ very rapidly, on a subsecond time scale. This enables them to accelerate the falling phase of the flash response and to modulate flash sensitivity during continuous illumination. To the contrary, the acceleration of transducin GTPase, even after significant reduction in cGMP, occurs over several tens of seconds. It is substantially delayed by the slow dissociation of cGMP from the noncatalytic sites for cGMP binding located on cGMP phosphodiesterase. Therefore, cGMP-dependent regulation of transducin GTPase is likely to occur only during prolonged bright illumination.

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Ca2+ dependence of frog ROS guanylate cyclase activity. The cyclase reaction was initiated by adding 10 μl of pseudointracellular medium containing 2 mM [γ-33P]GTP, 20 μM ATP, 20  mM [3H]-cGMP, and Ca2+ buffered to indicated concentration to  10 μl of a ROS suspension containing 40 μM rhodopsin and 200  μM zaprinast buffered to the same Ca2+. After 30 s, the reaction  was stopped with the addition of 80 μl of a quench solution containing 50 mM EDTA, pH 7.0. The cyclase activity is expressed as  μM cGMP produced in the equivalent ROS cytoplasm per second  and is plotted against the free Ca2+ concentration. Each point represents the mean ± SD of four separate determinations. The solid  line is a fit of Eq. 1 to the data with αmax = 9.74, αmin = 1.88, K1/2 =  256 nM Ca2+, and n = 1.82. (inset) A similar experiment carried  out at 180 μM rhodopsin. Here, fitting Eq. 1 to the data gave αmax =  12.7, αmin = 1.8, K1/2 = 230 nM Ca2+, and n = 1.85.
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Figure 1: Ca2+ dependence of frog ROS guanylate cyclase activity. The cyclase reaction was initiated by adding 10 μl of pseudointracellular medium containing 2 mM [γ-33P]GTP, 20 μM ATP, 20 mM [3H]-cGMP, and Ca2+ buffered to indicated concentration to 10 μl of a ROS suspension containing 40 μM rhodopsin and 200 μM zaprinast buffered to the same Ca2+. After 30 s, the reaction was stopped with the addition of 80 μl of a quench solution containing 50 mM EDTA, pH 7.0. The cyclase activity is expressed as μM cGMP produced in the equivalent ROS cytoplasm per second and is plotted against the free Ca2+ concentration. Each point represents the mean ± SD of four separate determinations. The solid line is a fit of Eq. 1 to the data with αmax = 9.74, αmin = 1.88, K1/2 = 256 nM Ca2+, and n = 1.82. (inset) A similar experiment carried out at 180 μM rhodopsin. Here, fitting Eq. 1 to the data gave αmax = 12.7, αmin = 1.8, K1/2 = 230 nM Ca2+, and n = 1.85.

Mentions: The regulation of guanylate cyclase activity by Ca2+ in rod photoreceptor outer segments has been well characterized in bovine rod photoreceptors (Koch and Stryer, 1988; Gorczyca et al., 1994b; Dizhoor et al., 1994). Ca2+ regulation of the guanylate cyclase in frog ROS has also been established (Coccia and Cote, 1994); however, a detailed analysis of the Ca2+ dependence was not performed. Such an analysis in a suspension of frog ROS is shown in Fig. 1. The cyclase activity was expressed as the cGMP concentration produced in the ROS cytoplasm per second. The value of 6 mM rhodopsin with respect to the ROS cytoplasm was used (see Bownds and Arshavsky, 1995). The Ca2+ dependence was approximated by the Hill equation: 1\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}{\alpha}={\alpha}_{max}-({\alpha}_{max}-{\alpha}_{min}){\cdot}\frac{Ca^{n}}{Ca^{n}+K_{1/2}^{n}}\end{equation*}\end{document}


Onset of feedback reactions underlying vertebrate rod photoreceptor light adaptation.

Calvert PD, Ho TW, LeFebvre YM, Arshavsky VY - J. Gen. Physiol. (1998)

Ca2+ dependence of frog ROS guanylate cyclase activity. The cyclase reaction was initiated by adding 10 μl of pseudointracellular medium containing 2 mM [γ-33P]GTP, 20 μM ATP, 20  mM [3H]-cGMP, and Ca2+ buffered to indicated concentration to  10 μl of a ROS suspension containing 40 μM rhodopsin and 200  μM zaprinast buffered to the same Ca2+. After 30 s, the reaction  was stopped with the addition of 80 μl of a quench solution containing 50 mM EDTA, pH 7.0. The cyclase activity is expressed as  μM cGMP produced in the equivalent ROS cytoplasm per second  and is plotted against the free Ca2+ concentration. Each point represents the mean ± SD of four separate determinations. The solid  line is a fit of Eq. 1 to the data with αmax = 9.74, αmin = 1.88, K1/2 =  256 nM Ca2+, and n = 1.82. (inset) A similar experiment carried  out at 180 μM rhodopsin. Here, fitting Eq. 1 to the data gave αmax =  12.7, αmin = 1.8, K1/2 = 230 nM Ca2+, and n = 1.85.
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Figure 1: Ca2+ dependence of frog ROS guanylate cyclase activity. The cyclase reaction was initiated by adding 10 μl of pseudointracellular medium containing 2 mM [γ-33P]GTP, 20 μM ATP, 20 mM [3H]-cGMP, and Ca2+ buffered to indicated concentration to 10 μl of a ROS suspension containing 40 μM rhodopsin and 200 μM zaprinast buffered to the same Ca2+. After 30 s, the reaction was stopped with the addition of 80 μl of a quench solution containing 50 mM EDTA, pH 7.0. The cyclase activity is expressed as μM cGMP produced in the equivalent ROS cytoplasm per second and is plotted against the free Ca2+ concentration. Each point represents the mean ± SD of four separate determinations. The solid line is a fit of Eq. 1 to the data with αmax = 9.74, αmin = 1.88, K1/2 = 256 nM Ca2+, and n = 1.82. (inset) A similar experiment carried out at 180 μM rhodopsin. Here, fitting Eq. 1 to the data gave αmax = 12.7, αmin = 1.8, K1/2 = 230 nM Ca2+, and n = 1.85.
Mentions: The regulation of guanylate cyclase activity by Ca2+ in rod photoreceptor outer segments has been well characterized in bovine rod photoreceptors (Koch and Stryer, 1988; Gorczyca et al., 1994b; Dizhoor et al., 1994). Ca2+ regulation of the guanylate cyclase in frog ROS has also been established (Coccia and Cote, 1994); however, a detailed analysis of the Ca2+ dependence was not performed. Such an analysis in a suspension of frog ROS is shown in Fig. 1. The cyclase activity was expressed as the cGMP concentration produced in the ROS cytoplasm per second. The value of 6 mM rhodopsin with respect to the ROS cytoplasm was used (see Bownds and Arshavsky, 1995). The Ca2+ dependence was approximated by the Hill equation: 1\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}{\alpha}={\alpha}_{max}-({\alpha}_{max}-{\alpha}_{min}){\cdot}\frac{Ca^{n}}{Ca^{n}+K_{1/2}^{n}}\end{equation*}\end{document}

Bottom Line: Light adaptation in vertebrate photoreceptors is thought to be mediated through a number of biochemical feedback reactions that reduce the sensitivity of the photoreceptor and accelerate the kinetics of the photoresponse.Guanylate cyclase activity and rhodopsin phosphorylation respond to changes in Ca2+ very rapidly, on a subsecond time scale.Therefore, cGMP-dependent regulation of transducin GTPase is likely to occur only during prolonged bright illumination.

View Article: PubMed Central - PubMed

Affiliation: Howe Laboratory of Ophthalmology, Harvard Medical School and the Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114, USA. pdcalvert@meei.harvard.edu

ABSTRACT
Light adaptation in vertebrate photoreceptors is thought to be mediated through a number of biochemical feedback reactions that reduce the sensitivity of the photoreceptor and accelerate the kinetics of the photoresponse. Ca2+ plays a major role in this process by regulating several components of the phototransduction cascade. Guanylate cyclase and rhodopsin kinase are suggested to be the major sites regulated by Ca2+. Recently, it was proposed that cGMP may be another messenger of light adaptation since it is able to regulate the rate of transducin GTPase and thus the lifetime of activated cGMP phosphodiesterase. Here we report measurements of the rates at which the changes in Ca2+ and cGMP are followed by the changes in the rates of corresponding enzymatic reactions in frog rod outer segments. Our data indicate that there is a temporal hierarchy among reactions that underlie light adaptation. Guanylate cyclase activity and rhodopsin phosphorylation respond to changes in Ca2+ very rapidly, on a subsecond time scale. This enables them to accelerate the falling phase of the flash response and to modulate flash sensitivity during continuous illumination. To the contrary, the acceleration of transducin GTPase, even after significant reduction in cGMP, occurs over several tens of seconds. It is substantially delayed by the slow dissociation of cGMP from the noncatalytic sites for cGMP binding located on cGMP phosphodiesterase. Therefore, cGMP-dependent regulation of transducin GTPase is likely to occur only during prolonged bright illumination.

Show MeSH
Related in: MedlinePlus