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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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Time window for the onset of fast transducin GTPase  after bright illumination. Suspensions of bleached ROS (30 μM  rhodopsin) were preincubated for 1 min with 3 μM [3H]cGMP.  cGMP dissociation from the PDE noncatalytic sites was initiated at  time zero by a chase with CaM/CaM-PDE (80,000 U/liter CaM,  1,000 U/liter CaM-PDE) alone (▿), CaM/CaM-PDE with 20 μM  GTPγS (○), or with 20 μM GTPγS alone (•). The amounts of  [3H]cGMP bound to PDE were determined as described previously. A single exponential process was fit to the data for nonactivated PDE yielding a rate constant of 0.0033 s−1. The data for the  dissociation of cGMP from activated PDE with and without the  CaM/CaM-PDE chase were each approximated with the sum of  two exponential processes. In the case where CaM/CaM-PDE was  included in the chase, cGMP dissociated from 33% of the sites with  a rate constant of 0.170 s−1 and from 67% of the sites with a rate  constant of 0.0082. In the case where the chase was initiated with  GTPγS alone, cGMP dissociated from 33% of the sites with a rate  constant of 0.095 s−1 and from 67% of the sites with a rate constant  of 0.0058 s−1. The figure is representative of three similar experiments.
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Figure 7: Time window for the onset of fast transducin GTPase after bright illumination. Suspensions of bleached ROS (30 μM rhodopsin) were preincubated for 1 min with 3 μM [3H]cGMP. cGMP dissociation from the PDE noncatalytic sites was initiated at time zero by a chase with CaM/CaM-PDE (80,000 U/liter CaM, 1,000 U/liter CaM-PDE) alone (▿), CaM/CaM-PDE with 20 μM GTPγS (○), or with 20 μM GTPγS alone (•). The amounts of [3H]cGMP bound to PDE were determined as described previously. A single exponential process was fit to the data for nonactivated PDE yielding a rate constant of 0.0033 s−1. The data for the dissociation of cGMP from activated PDE with and without the CaM/CaM-PDE chase were each approximated with the sum of two exponential processes. In the case where CaM/CaM-PDE was included in the chase, cGMP dissociated from 33% of the sites with a rate constant of 0.170 s−1 and from 67% of the sites with a rate constant of 0.0082. In the case where the chase was initiated with GTPγS alone, cGMP dissociated from 33% of the sites with a rate constant of 0.095 s−1 and from 67% of the sites with a rate constant of 0.0058 s−1. The figure is representative of three similar experiments.

Mentions: Having established that the increase in transducin GTPase directly follows the dissociation of cGMP from the PDE noncatalytic sites, we wanted to know how fast cGMP might dissociate from these sites during the photoreceptor light response. The dissociation rate derived from the data presented in Fig. 5 gives only a slower limit for this process because PDE activation by transducin results in an accelerated rate of cGMP dissociation from the noncatalytic sites (Yamazaki et al., 1982, 1996; Cote et al., 1994). In Fig. 7, we compare the rates of cGMP dissociation from the noncatalytic sites of activated and nonactivated PDE after a chase with CaM/CaM-PDE. As reported earlier (Cote and Brunnock, 1993; Cote et al., 1994; Yamazaki et al., 1996) and as shown above in Fig. 5, cGMP dissociation from nonactivated PDE is a single exponential process, while cGMP dissociation from transducin-activated PDE is biphasic. cGMP dissociates from 32 ± 7% of the sites with a rate constant of 0.11 ± 0.04 s−1 and from 68 ± 7% with a rate constant of 0.006 ± 0.001 s−1 (n = 5). These rates are at least threefold faster than those obtained by Cote et al. (1994) after a chase with an excess of unlabeled cGMP. A reasonable explanation for this difference is provided by Yamazaki et al. (1996), who suggested that cGMP release from the noncatalytic sites of activated PDE may be inhibited by high concentrations of cGMP. Cote et al. (1994) and Yamazaki et al. (1996) proposed that the biphasic cGMP dissociation is due to a heterogeneity in the noncatalytic cGMP binding sites. However, the mechanism may be different considering that the maximal high affinity cGMP binding observed in our experiments corresponds to two cGMP molecules per PDE holoenzyme, while the amplitudes of the two phases of cGMP dissociation are twofold different rather than being equal as expected.


Onset of feedback reactions underlying vertebrate rod photoreceptor light adaptation.

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

Time window for the onset of fast transducin GTPase  after bright illumination. Suspensions of bleached ROS (30 μM  rhodopsin) were preincubated for 1 min with 3 μM [3H]cGMP.  cGMP dissociation from the PDE noncatalytic sites was initiated at  time zero by a chase with CaM/CaM-PDE (80,000 U/liter CaM,  1,000 U/liter CaM-PDE) alone (▿), CaM/CaM-PDE with 20 μM  GTPγS (○), or with 20 μM GTPγS alone (•). The amounts of  [3H]cGMP bound to PDE were determined as described previously. A single exponential process was fit to the data for nonactivated PDE yielding a rate constant of 0.0033 s−1. The data for the  dissociation of cGMP from activated PDE with and without the  CaM/CaM-PDE chase were each approximated with the sum of  two exponential processes. In the case where CaM/CaM-PDE was  included in the chase, cGMP dissociated from 33% of the sites with  a rate constant of 0.170 s−1 and from 67% of the sites with a rate  constant of 0.0082. In the case where the chase was initiated with  GTPγS alone, cGMP dissociated from 33% of the sites with a rate  constant of 0.095 s−1 and from 67% of the sites with a rate constant  of 0.0058 s−1. The figure is representative of three similar experiments.
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Related In: Results  -  Collection

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Figure 7: Time window for the onset of fast transducin GTPase after bright illumination. Suspensions of bleached ROS (30 μM rhodopsin) were preincubated for 1 min with 3 μM [3H]cGMP. cGMP dissociation from the PDE noncatalytic sites was initiated at time zero by a chase with CaM/CaM-PDE (80,000 U/liter CaM, 1,000 U/liter CaM-PDE) alone (▿), CaM/CaM-PDE with 20 μM GTPγS (○), or with 20 μM GTPγS alone (•). The amounts of [3H]cGMP bound to PDE were determined as described previously. A single exponential process was fit to the data for nonactivated PDE yielding a rate constant of 0.0033 s−1. The data for the dissociation of cGMP from activated PDE with and without the CaM/CaM-PDE chase were each approximated with the sum of two exponential processes. In the case where CaM/CaM-PDE was included in the chase, cGMP dissociated from 33% of the sites with a rate constant of 0.170 s−1 and from 67% of the sites with a rate constant of 0.0082. In the case where the chase was initiated with GTPγS alone, cGMP dissociated from 33% of the sites with a rate constant of 0.095 s−1 and from 67% of the sites with a rate constant of 0.0058 s−1. The figure is representative of three similar experiments.
Mentions: Having established that the increase in transducin GTPase directly follows the dissociation of cGMP from the PDE noncatalytic sites, we wanted to know how fast cGMP might dissociate from these sites during the photoreceptor light response. The dissociation rate derived from the data presented in Fig. 5 gives only a slower limit for this process because PDE activation by transducin results in an accelerated rate of cGMP dissociation from the noncatalytic sites (Yamazaki et al., 1982, 1996; Cote et al., 1994). In Fig. 7, we compare the rates of cGMP dissociation from the noncatalytic sites of activated and nonactivated PDE after a chase with CaM/CaM-PDE. As reported earlier (Cote and Brunnock, 1993; Cote et al., 1994; Yamazaki et al., 1996) and as shown above in Fig. 5, cGMP dissociation from nonactivated PDE is a single exponential process, while cGMP dissociation from transducin-activated PDE is biphasic. cGMP dissociates from 32 ± 7% of the sites with a rate constant of 0.11 ± 0.04 s−1 and from 68 ± 7% with a rate constant of 0.006 ± 0.001 s−1 (n = 5). These rates are at least threefold faster than those obtained by Cote et al. (1994) after a chase with an excess of unlabeled cGMP. A reasonable explanation for this difference is provided by Yamazaki et al. (1996), who suggested that cGMP release from the noncatalytic sites of activated PDE may be inhibited by high concentrations of cGMP. Cote et al. (1994) and Yamazaki et al. (1996) proposed that the biphasic cGMP dissociation is due to a heterogeneity in the noncatalytic cGMP binding sites. However, the mechanism may be different considering that the maximal high affinity cGMP binding observed in our experiments corresponds to two cGMP molecules per PDE holoenzyme, while the amplitudes of the two phases of cGMP dissociation are twofold different rather than being equal as expected.

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