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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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The extent of Ca2+-dependent regulation of guanylate  cyclase activity in frog ROS increases with the increase in ROS concentration. (A) The cyclase activity in ROS suspensions containing  indicated amounts of rhodopsin was measured at 13 nM (○) and  13 μM (•) Ca2+ as described for Fig. 1, except that 1 mM GTP was  used and the incubation time was 20 s. The lines represent linear  regression drawn through the points. (B) The ratios of values obtained in A for low and high Ca2+ are plotted as a function of ROS  concentration. The line is a linear regression drawn through the  points. All data are taken from one of three identical experiments.
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Figure 2: The extent of Ca2+-dependent regulation of guanylate cyclase activity in frog ROS increases with the increase in ROS concentration. (A) The cyclase activity in ROS suspensions containing indicated amounts of rhodopsin was measured at 13 nM (○) and 13 μM (•) Ca2+ as described for Fig. 1, except that 1 mM GTP was used and the incubation time was 20 s. The lines represent linear regression drawn through the points. (B) The ratios of values obtained in A for low and high Ca2+ are plotted as a function of ROS concentration. The line is a linear regression drawn through the points. All data are taken from one of three identical experiments.

Mentions: In the next set of experiments, we determined whether the parameters of the Ca2+-dependent cyclase regulation are dependent on the ROS concentration used in the assay. While K1/2 and n are essentially unaffected, the magnitude of the effect is higher at high than at low ROS concentrations (see Fig. 1, inset). A more thorough investigation of the ROS concentration effect on the extent of cyclase regulation by Ca2+ is shown in Fig. 2, where the cyclase rates at low and high Ca2+ were measured at various concentrations of ROS taken from the same stock. The increase in ROS concentration is accompanied by both an increase of cyclase activity at low Ca2+ and a decrease in cyclase activity at high Ca2+ (Fig. 2 A). This result is consistent with a larger fraction of cyclase being in a complex with guanylate cyclase–activating protein, which, according to Dizhoor and Hurley (1996), serves both as cyclase activator at low Ca2+ and cyclase inhibitor at high Ca2+. As shown in Fig. 2 B, the highest ratio that we observed was ∼10-fold. This effect does not saturate over the range of ROS concentration that we could study, precluding us from providing the exact magnitude of this regulation in intact frog ROS.


Onset of feedback reactions underlying vertebrate rod photoreceptor light adaptation.

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

The extent of Ca2+-dependent regulation of guanylate  cyclase activity in frog ROS increases with the increase in ROS concentration. (A) The cyclase activity in ROS suspensions containing  indicated amounts of rhodopsin was measured at 13 nM (○) and  13 μM (•) Ca2+ as described for Fig. 1, except that 1 mM GTP was  used and the incubation time was 20 s. The lines represent linear  regression drawn through the points. (B) The ratios of values obtained in A for low and high Ca2+ are plotted as a function of ROS  concentration. The line is a linear regression drawn through the  points. All data are taken from one of three identical experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1887766&req=5

Figure 2: The extent of Ca2+-dependent regulation of guanylate cyclase activity in frog ROS increases with the increase in ROS concentration. (A) The cyclase activity in ROS suspensions containing indicated amounts of rhodopsin was measured at 13 nM (○) and 13 μM (•) Ca2+ as described for Fig. 1, except that 1 mM GTP was used and the incubation time was 20 s. The lines represent linear regression drawn through the points. (B) The ratios of values obtained in A for low and high Ca2+ are plotted as a function of ROS concentration. The line is a linear regression drawn through the points. All data are taken from one of three identical experiments.
Mentions: In the next set of experiments, we determined whether the parameters of the Ca2+-dependent cyclase regulation are dependent on the ROS concentration used in the assay. While K1/2 and n are essentially unaffected, the magnitude of the effect is higher at high than at low ROS concentrations (see Fig. 1, inset). A more thorough investigation of the ROS concentration effect on the extent of cyclase regulation by Ca2+ is shown in Fig. 2, where the cyclase rates at low and high Ca2+ were measured at various concentrations of ROS taken from the same stock. The increase in ROS concentration is accompanied by both an increase of cyclase activity at low Ca2+ and a decrease in cyclase activity at high Ca2+ (Fig. 2 A). This result is consistent with a larger fraction of cyclase being in a complex with guanylate cyclase–activating protein, which, according to Dizhoor and Hurley (1996), serves both as cyclase activator at low Ca2+ and cyclase inhibitor at high Ca2+. As shown in Fig. 2 B, the highest ratio that we observed was ∼10-fold. This effect does not saturate over the range of ROS concentration that we could study, precluding us from providing the exact magnitude of this regulation in intact frog ROS.

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