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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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cGMP dissociation  from the PDE noncatalytic sites  after chase with CaM/CaM-PDE  (A) or excess cGMP (B). Suspensions of frog ROS (30 μM  rhodopsin) were preincubated  for 1 min in pseudointracellular  medium containing 50 μM Ca2+  and 3 μM [3H]cGMP, and then  [3H]cGMP dissociation from the  PDE noncatalytic sites was initiated at time zero by a chase with  either CaM/CaM-PDE (80 activity units CaM per unit CaM-PDE)  or 2 mM nonlabeled cGMP. The  amounts of [3H]cGMP bound to  PDE were determined as described in methods. CaM-PDE  concentrations (U/liter): •,  none; □, 7.5; ▪, 37; ▴, 150; ○, 740. The data from two lower curves of A and the curve in B are single exponential fits. The rest of the  curves are hand drawn. The figure is representative of three similar experiments.
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Figure 5: cGMP dissociation from the PDE noncatalytic sites after chase with CaM/CaM-PDE (A) or excess cGMP (B). Suspensions of frog ROS (30 μM rhodopsin) were preincubated for 1 min in pseudointracellular medium containing 50 μM Ca2+ and 3 μM [3H]cGMP, and then [3H]cGMP dissociation from the PDE noncatalytic sites was initiated at time zero by a chase with either CaM/CaM-PDE (80 activity units CaM per unit CaM-PDE) or 2 mM nonlabeled cGMP. The amounts of [3H]cGMP bound to PDE were determined as described in methods. CaM-PDE concentrations (U/liter): •, none; □, 7.5; ▪, 37; ▴, 150; ○, 740. The data from two lower curves of A and the curve in B are single exponential fits. The rest of the curves are hand drawn. The figure is representative of three similar experiments.

Mentions: The kinetics of cGMP dissociation from the noncatalytic sites of nonactivated PDE after addition of various amounts of CaM/CaM-PDE are shown in Fig. 5 A. A suspension of frog ROS was preincubated with 3 μM [3H]cGMP for 1 min, and then, at time zero, either buffer (Fig. 5 A, upper curve) or indicated amounts of CaM/CaM-PDE were added. The addition of buffer alone causes a very slow reduction in the amount of [3H]cGMP bound to the PDE noncatalytic sites. This process is slow most likely because the probability of dissociated cGMP to rebind with the noncatalytic sites is higher than the probability for this cGMP to be hydrolyzed by the basal PDE activity. Additions of increasing concentrations of CaM/CaM-PDE result in a faster decline of the amount of [3H]cGMP bound to the PDE noncatalytic sites until saturation occurs at CaM-PDE concentration of ∼150 activity U/liter. cGMP dissociation at saturating concentrations of CaM-PDE is described as a single exponential process with a rate constant of 0.0032 ± 0.0003 s−1 (n = 8), close to the rate constant observed after a chase with excess nonlabeled cGMP (0.0025 ± 0.0002 s−1, n = 3; Fig. 5 B).


Onset of feedback reactions underlying vertebrate rod photoreceptor light adaptation.

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

cGMP dissociation  from the PDE noncatalytic sites  after chase with CaM/CaM-PDE  (A) or excess cGMP (B). Suspensions of frog ROS (30 μM  rhodopsin) were preincubated  for 1 min in pseudointracellular  medium containing 50 μM Ca2+  and 3 μM [3H]cGMP, and then  [3H]cGMP dissociation from the  PDE noncatalytic sites was initiated at time zero by a chase with  either CaM/CaM-PDE (80 activity units CaM per unit CaM-PDE)  or 2 mM nonlabeled cGMP. The  amounts of [3H]cGMP bound to  PDE were determined as described in methods. CaM-PDE  concentrations (U/liter): •,  none; □, 7.5; ▪, 37; ▴, 150; ○, 740. The data from two lower curves of A and the curve in B are single exponential fits. The rest of the  curves are hand drawn. The figure is representative of three similar experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1887766&req=5

Figure 5: cGMP dissociation from the PDE noncatalytic sites after chase with CaM/CaM-PDE (A) or excess cGMP (B). Suspensions of frog ROS (30 μM rhodopsin) were preincubated for 1 min in pseudointracellular medium containing 50 μM Ca2+ and 3 μM [3H]cGMP, and then [3H]cGMP dissociation from the PDE noncatalytic sites was initiated at time zero by a chase with either CaM/CaM-PDE (80 activity units CaM per unit CaM-PDE) or 2 mM nonlabeled cGMP. The amounts of [3H]cGMP bound to PDE were determined as described in methods. CaM-PDE concentrations (U/liter): •, none; □, 7.5; ▪, 37; ▴, 150; ○, 740. The data from two lower curves of A and the curve in B are single exponential fits. The rest of the curves are hand drawn. The figure is representative of three similar experiments.
Mentions: The kinetics of cGMP dissociation from the noncatalytic sites of nonactivated PDE after addition of various amounts of CaM/CaM-PDE are shown in Fig. 5 A. A suspension of frog ROS was preincubated with 3 μM [3H]cGMP for 1 min, and then, at time zero, either buffer (Fig. 5 A, upper curve) or indicated amounts of CaM/CaM-PDE were added. The addition of buffer alone causes a very slow reduction in the amount of [3H]cGMP bound to the PDE noncatalytic sites. This process is slow most likely because the probability of dissociated cGMP to rebind with the noncatalytic sites is higher than the probability for this cGMP to be hydrolyzed by the basal PDE activity. Additions of increasing concentrations of CaM/CaM-PDE result in a faster decline of the amount of [3H]cGMP bound to the PDE noncatalytic sites until saturation occurs at CaM-PDE concentration of ∼150 activity U/liter. cGMP dissociation at saturating concentrations of CaM-PDE is described as a single exponential process with a rate constant of 0.0032 ± 0.0003 s−1 (n = 8), close to the rate constant observed after a chase with excess nonlabeled cGMP (0.0025 ± 0.0002 s−1, n = 3; Fig. 5 B).

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