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1 From the Department of Ophthalmology, University Eye Clinic, Zurich, Switzerland; and 2 Department of Biological Science, Florida State University, Tallahassee.
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Abstract |
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METHODS. Eyes of anesthetized albino rats were exposed to either green (550 nm) or deep blue (403 nm) light, and the time course of rhodopsin bleaching was determined. Rhodopsin was isolated from whole retinas by detergent extraction and measured photometrically. To inhibit photoreversal of bleaching, rats were perfused with 70 mM hydroxylamine (NH2OH), a known inhibitor of photoreversal. To determine whether blue-absorbing, photoreversible photoproducts were formed, rhodopsin was bleached to near completion with green light and then exposed to blue light. Finally, experimental results were simulated on a computer by means of a simple, three-component model involving a long-lived photoreversible photoproduct.
RESULTS. Photoreversal of bleaching in blue light occurs in vivo as evidenced by the following: In the absence of NH2OH, bleaching of rhodopsin by blue light was slow and complex. In the presence of NH2OH, however, blue light bleached rhodopsin very fast with a simple, pseudo–first-order kinetic. A long-lived bleaching intermediate produced by green light exposure was photoreversed to rhodopsin by exposure to blue light. The three-component computer model, invoking a blue-absorbing, photoreversible, long-lived intermediate accurately described the data.
CONCLUSIONS. Because of the instantaneous, nonmetabolic regeneration of rhodopsin by the process of photoreversal of bleaching, blue light exposure permits the absorption of large numbers of photons by rhodopsin and by a photoreversible intermediate of bleaching in vivo. These data may have an important impact on resolving mechanisms of blue light–mediated damage to the retina.
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Introduction |
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The visual pigment rhodopsin consists of 11-cis-retinal and opsin, the apoprotein of the pigment. Photon absorption causes bleaching of rhodopsin, a process that is initiated by the photoconversion of 11-cis-retinal to all-trans-retinal. Subsequently, rhodopsin undergoes a series of dark reactions that culminate in the dissociation of retinal, thus completing the process of bleaching. However, before this dissociation occurs, an intermediate of the bleaching process, metarhodopsin II (MII),4 5 interacts with the G-protein transducin. This interaction initiates a cyclic nucleotide cascade that ultimately converts the absorbed light into an electrical response of the rod cell. Quenching of G-protein activation occurs by binding of arrestin to MII.6 Because the absorption maximum of the bleaching intermediate MII is at 380 nm,7 MII is spectrophotometrically virtually indistinguishable from dissociated, free all-trans-retinal, a truly bleached product.7 8 9 However, a significant difference between free retinal and MII is that, in MII, the all-trans-retinal continues to occupy its chromophoric site in opsin, whereas free retinal is not attached to opsin.8 Furthermore, the all-trans-retinal transiently binds to phosphatidyl-ethanolamine and, after reduction to all-trans-retinol, is transported to the pigmented epithelium.10 11
Hubbard and Kropf12 discovered that normal photobleaching could be prevented if additional light was absorbed by bleaching intermediates. This prevention of bleaching is effected by a trans-to-cis photograph reisomerization of the retinal chromophore after absorption of additional photons, and the process was thus called "photoreversal" of bleaching.13 That photoregeneration created functional rhodopsin from bleaching intermediates was also suggested by Cone, who reported that a complete bleaching of rhodopsin abolishes early receptor potentials in excised rat eyes, but that they can be recorded again after blue light is flashed onto the eye. Thus, rhodopsin must have been restored by the absorption of the additional light of short wavelength.14 More light absorbed caused less bleaching. Indeed, working at low temperatures to extend the lifetime of successive intermediates, Hubbard and Kropf12 showed that photograph steady states between a given intermediate and a mixture of rhodopsin and isorhodopsin (9-cis-chromophore) were possible and were indefinitely stable as long as the temperature was maintained. That is, the system was "closed." They further showed that the quantum efficiencies for the cis-trans and trans-to-cis reactions were virtually identical (0.7) for the earliest intermediates. A critical stage of bleaching arose when MII appeared during irradiation. Suddenly, less rhodopsin could be photoregenerated, and the maximum amount of rhodopsin bleached by a flash increased dramatically. Although the system became "leaky," it was nevertheless concluded that the quantum efficiency for photoconverting MII to stable, light-sensitive pigments was lower than that of the earlier intermediates, but that it was not zero.4 15 16 17
It can be deduced from the work of Hubbard and Kropf12 that three conditions are necessary for maintaining photograph steady states between rhodopsin and any given intermediate of bleaching: The intermediate absorbing the additional light must retain retinal at the chromophoric site, the incoming photons must be of the proper wavelength to be absorbed by that intermediate, and the intermediate absorbing the light must have a lifetime that is longer than or at least commensurate with the rate at which photons are coming in (i.e., intensity) and being absorbed.
Except for the flash photolysis study by Hagins18 and the observations of Cone,14 all the work referenced thus far was performed in vitro. In the present study we show by direct measurements of the course of bleaching of rhodopsin that blue light photoregenerated a relatively long-lived, blue-absorbing intermediate in vivo. Based on our results and the identification of rhodopsin as chromophore for light damage,1 2 3 we show that in experiments of blue light damage,19 20 21 22 23 24 photoreversal of bleaching may have occurred, leading to the absorption of high numbers of photons per unit of time and rendering blue light highly effective in the mediation of light damage.
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Methods |
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Light Exposure
Left eyes of anesthetized rats (ketamine 75 mg/kg and xylazine 23
mg/kg) were kept moist with Methocel (CibaVision, Hergiswil,
Switzerland) and exposed either to deep blue light (403 ± 10 nm)
or to green light (550 ± 10 nm) for up to 30 minutes. The light
exposure system consisted of a xenon short-arc reflector lamp (230 V,
50 Hz, 120 W; Intralux MDR 100; Volpi, Schlieren, Switzerland) with
interference filters to eliminate UV and infrared radiation and
a liquid fiberoptic light guide (8 mm in diameter) to the animal’s
eye. The optical system included a switch holder for blue (403 ±
10 nm, bandwidth) or green (550 ± 10 nm bandwidth) interference
filters.
Intensities of exposure were at 300 µW/cm2 for 403-nm blue light and 409 µW/cm2 for 550-nm green light. These intensities gave equal photon fluxes at the cornea of 6 x 1014/sec per square centimeter.
NH2OH Studies: Inhibition of Photoreversal
NH2OH actively removes the retinal from the
chromophoric site of late-bleaching intermediates including
MII,25
26
without affecting rhodopsin itself (data not
shown).27
28
29
Although inhibition of photoreversal of
bleaching by NH2OH is very efficient, it may
nevertheless be incomplete: some intermediates may still be able to
absorb enough photons to be photoreversed before
NH2OH can remove retinal from the chromophoric
site in opsin, which will, perforce, prevent photoreversal of
bleaching. To apply NH2OH, deeply anesthetized
animals were cardially perfused with NH2OH
solution (70 mM in 0.9% NaCl [pH 7.2], 100 ml) in dim red light
immediately before irradiation of the eye with blue light, as described
earlier.
Rhodopsin Measurements and Calculation of the First-Order Rate
Constants
Retinas were extruded through slits across the corneas and
immersed in 1 ml distilled water for 30 seconds. After centrifugation
at 14,000g for 3 minutes at room temperature, supernatants
were discarded, and 0.7 ml cetyltrimethylammonium bromide (CTAB, 1% in
H2O) was added to each retina pellet. Retinas
were homogenized with a polytron for 20 seconds, and the homogenates
were centrifuged at 14,000g for 3 minutes (room
temperature). Supernatants were drawn off quantitatively and scanned on
a rapid-recording spectrophotometer (Carry 50; Varian, Basel,
Switzerland). After scanning, rhodopsin was bleached completely by
exposing the retinal extract to 20,000 lux of white light for 1 minute.
Extracts were scanned again, and the difference in absorbancy of
rhodopsin at 500 nm was determined. This value was used to calculate
from the Beer-Lambert law (molar absorbance coefficient = 42,000
M/cm) the concentration of rhodopsin in the extract, as described
recently.30
Because the intensity was held constant during a run, the rate of bleaching (Kb) is a first-order rate constant and has units of (time-1). Thus, Kb = ln (Rt/R0)/t; where (Rt/R0) is the fraction of rhodopsin remaining after exposure during a time (t).
Computer Simulation of Results
The experimental results were simulated on a computer (Excel;
Microsoft, Redmond, WA). The authors will supply details of this
simulation on request. The model simulated was
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Results |
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Although long-wave green light does not cause photoreversal of bleaching (see above),32 33 rhodopsin was not bleached to completion. When rhodopsin was bleached using a 5-minute green flash (550 nm; 409 µW/cm2), rhodopsin was metabolically regenerated to 13% of the dark value during 15 minutes in darkness (data not shown). Therefore, the metabolic regeneration of rhodopsin in the visual cycle may have restored enough rhodopsin during exposure that bleaching and regeneration reached an equilibrium at very low levels (less than 5%).
Photoregeneration of Rhodopsin by Blue Light after Green Light
Bleaching
Green light bleaches rhodopsin without causing photoreversal (see
above).32
33
After 5 minutes of exposure to green light,
less than 5% of rhodopsin was left (Fig. 2)
. Continuation of the green light exposure decreased this value even
further. However, when the green light (after 5 minutes) was rapidly
switched to blue light (for 1 minute), rhodopsin was regenerated to
28% on average (Fig. 2)
. Obviously, some intermediates of the green
light–induced bleaching process were long lived and capable of
reversal to rhodopsin after absorption of additional blue light. More
light appeared to cause less bleaching, a hallmark of photoreversal of
bleaching.
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Of interest is that the lines were straight over 2 and 3 ln units (Fig. 3 ; r2 > 0.99 for both). This implies that photoreversal is virtually completely avoided in green light and inhibited by NH2OH in blue light. If photoreversal were to occur, as in the bleaching process with blue light in the absence of NH2OH, the lines would be curvilinear—the more photoregeneration, the greater the curvature. This is further supported by direct measurements of the rhodopsin remaining after a 1-minute green light bleaching in the absence or presence of NH2OH. The value was identical in both conditions (12%–13%) strongly suggesting that photoreversal of bleaching does not occur in long-wave green light (550 nm) or only very little so.
A Model to Determine Bleaching
Development of the model was based largely on the value of
Kb and on the shape of the blue
no-NH2OH curve in Figure 1
. Inspection of the
function without NH2OH shows that the curve
declined very slowly after an initial bleaching burst and that a
substantial amount of rhodopsin remained even after 30 minutes of
exposure. If bleaching depended only on rhodopsin and a photoreversible
intermediate, the system would be closed and the cis-trans
isomerizations (bleaching and photoregeneration) would come into steady
state, the level of which would be determined by the relative
photosensitivities of rhodopsin and the blue absorber. However, the
slow decline of the rhodopsin level in blue light indicated that the
system was not closed; it was leaky. Photoreversibility was gradually
lost, and a steady state could not be maintained, even though the light
intensity was held constant. This leak in the system was most likely
due to the slow decay of the photoreversible intermediates (MII) to
products (opsin and free all-trans-retinal, which is
converted rapidly to all-trans-retinol). The products enter
the visual cycle to regenerate rhodopsin physiologically through a
reisomerization–oxidation step of the all-trans-retinol in
the pigment epithelium.
The measured value of Kb and estimated values of Kpr and Kl were put into a set of differential rate equations as described in the Methods section, and the equations were solved simultaneously. The results of this simulation are given in Figure 4 , along with the experimentally determined data points of the blue bleach (in the absence of NH2OH, Fig. 1 ). With Kb fixed at the measured value of 0.61 per minute, the best fit values of Kpr and Kl were 0.35 per minute and 0.075 per minute, respectively. This suggests that trans-cis (photoreversal) absorptions at 403 nm occur approximately half as often as do cis-trans (bleaching) absorptions (Kpr/Kb = 0.35/0.61 = 0.57). Also, the calculated leak rate was approximately one fourth the photoreversal rate (Kl/Kpr = 0.075/0.35 = 0.21), large enough to prevent a true photograph steady state between rhodopsin and the blue absorber. The good fit of the measured data points to the calculated curve indicates that the model with only two photoconvertible states and one leak step is sufficient to describe the data.
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, where
Kb is the rate constant for rhodopsin
bleaching with NH2OH in blue light of 300
µW/cm2—namely, 0.61 per minute.
Rt is the rhodopsin present at any
time (t), and is the quantum efficiency with which
rhodopsin is bleached, independent of the wavelength (0.7 molecules
bleached/absorbed photon).12 In Table 1 we summarize the results of the calculations to determine the numbers of photons absorbed by rhodopsin in the presence and absence of NH2OH. We also present the numbers of photons absorbed by the blue absorber, assuming its time course is as given by the model (Fig. 4) and that it is MII. To obtain these numbers we integrated the curves graphically (and, for confirmation, by numerical means as well; data not shown) from t = 0 to t = 5, 10, and 30 minutes. The total number of absorptions by the average molecule (combining absorptions while in the rhodopsin state with those while in the MII state) during 30 minutes of blue light exposure (403 nm at 300 µW/cm2) was approximately 55. This is a surprisingly large amount of light absorbed by individual molecules.
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= 0.7,
1.4 (1/0.7 = 1.4) photons must be absorbed, on average, to bleach
one molecule. Similarly, the photoreversal of MII has a quantum
efficiency of 0.22 for producing one rhodopsin34
;
therefore, 4.5 (1/0.22 = 4.5) photons, on average, must be
absorbed by MII to produce one rhodopsin. Figure 5 shows an abbreviated scheme for conceptualizing bleaching and photoreversal in 403-nm light. To complete one bleaching and regeneration cycle, a rhodopsin molecule (and the reversible intermediate) must therefore absorb approximately six photons of blue light at the given wavelength and intensity. In the 30 minutes during which 55 absorptions occur, a single rhodopsin molecule completes on average approximately nine cycles of bleaching and regeneration.
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Discussion |
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Therefore, we conclude that the apparent slowness of rhodopsin bleaching by blue light, in the absence of NH2OH, is not due to a failure of rhodopsin to absorb that light. Rather, the slowness is a result of the rapid regeneration of the rhodopsin by the photograph reisomerization of the all-trans-retinal that resides in the chromophoric site of a long-lived photoproduct. Before bleached rhodopsin can absorb another photon, it usually must be regenerated in the visual cycle, a slow process (15 minutes in darkness regenerated rhodopsin to 13% of dark levels after a 5-minute green light bleaching; data not shown) that involves shuttling of the all-trans-retinol to the pigment epithelium, enzymatic reisomerization to 11-cis-retinol, and the transport back to the photoreceptors. In blue light conditions, regeneration can occur very fast through the process of photoreversal, and a single rhodopsin molecule can therefore absorb many photons and can enter the bleaching pathway several times in a short period.
Evidence suggests that the long-lived intermediate that is
photoreversed by blue light may be MII: (1) The retinal is still
attached to the chromophoric site in MII. Thus, any reisomerization of
the MII retinal to 11- or 9-cis occurs in the chromophoric
pocket of opsin and thus efficiently restores rhodopsin. (2) MII,
similar to retinal, has an absorption maximum of 380 nm,35
which is sufficiently close to the 403 nm used in this study. MII
absorbs light at 403 nm approximately 70% to 80% as efficiently as
rhodopsin does at 500 nm.36
Furthermore,
Williams34
found that in vitro 1 of 4.5 absorptions
converts MII to rhodopsin, and therefore the quantum efficiency of this
process (0.22) is high enough to photoconvert reasonable amounts of the
all-trans to 11- or 9-cis, even at 403 nm. (3)
MII is a long-lived bleaching intermediate with a lifetime measured in
seconds.37
Our light source projects a large number of
photons every second onto the cornea (
3 x
1015 photons/sec). Even though approximately 20%
of these photons (at 403 nm wavelength) are absorbed by the rat lens
and therefore do not reach the retina38
and a large
fraction of the photons is not absorbed by rhodopsin,31
such intensities are sufficient to "hit" substantial numbers of MII
molecules before they decay irreversibly.
Other deep blue–absorbing photoproducts can be ruled out as candidates for the reversible intermediate, even though some of them fulfill some of the necessary criteria. These molecules include retinal, retinol, N-retinylidene-N-retinylphosphatidyl-ethanolamine, N-retinylidene-N-retinylethanolamine,39 40 and N-retinylidene phosphatidylethanolamin.11 The spectra of these are appropriate for absorbing 403-nm light strongly. Of importance, however, is that the retinoid components of these molecules no longer occupy the chromophoric site on opsin, and they are therefore unlikely candidates for photoreversible molecules. Furthermore, after dissociation, retinal and retinol assume orientations in the disc membrane that are perpendicular to the disc. Thus, their absorption dipoles are orthogonal to the e vector of incoming light.41 42 They are therefore extremely weak absorbers of light in vivo. The orientations in vivo of the other three compounds have not been determined.
Another blue-absorbing candidate, metarhodopsin III (MIII,
max = 480 nm), should be considered separately
from the others, because there is discord about its photoreversibility.
Williams34
found that MIII was not photoreversible but
Mathews et al.8
reported that it was weakly and indirectly
(through MII) reversible with light. Even if weakly reversible, the
time course of MIII’s appearance does not fit our observations: Figure 1
shows that photoreversibility in the blue light was present early
during the irradiation. This is evidenced by the fact that the two blue
curves (with and without NH2OH)
diverged early—i.e., the difference in
bleachability appeared soon during the exposures. In addition, the
model (Fig. 4)
indicates that photoreversibility began to be lost after
approximately 1 minute as the product appeared. Taken together, these
facts strongly support the notion that the photoreversible intermediate
is MII, because MII appears within approximately a
millisecond4
13
15
34
and then begins a slow decay to MIII
(or retinal).8
9
If MIII were the photoreversible blue
absorber, photoreversibility would have increased in long time interval
as MIII appeared. Although we cannot completely exclude that a minor
amount of rhodopsin is regenerated from MIII, we nevertheless conclude,
by a process of elimination, that MII is the main reversible blue
absorber in the rat retina.
It is not clear what the molecular or cellular effects of so many photon absorptions by one molecule might be. It seems certain, however, that photoregenerated pigment can produce electrophysiological responses and thus constitutes functional rhodopsin.14 43 Beyond this, however, a number of questions remain unanswered. (1) Is MII photoreversible when either transducin or arrestin is attached, or must these ligands be dissociated? Arnis and Hofmann,4 using an excess of transducin, found in vitro that the binding to transducin inhibits reversal of the intermediate if no nucleotides are available, but it is not clear what happens in in vivo conditions. (2) What happens to transduction or to response shutoff if absorptions occur while MII is bound to these ligands? (3) Do some of the deleterious effects of blue light derive from interfering with these processes by trans-cis photoisomerization? Answers to such questions could help to elucidate whether damage in blue light is due simply to an excessive number of photon absorptions or to absorptions at certain time points (e.g., during transduction or response shutoff).
In conclusion, we have shown that the apparent slowness of rhodopsin bleaching in blue light is not due to a failure of rhodopsin to absorb that light44 but is caused by the photoreversal of bleaching. Conclusions about the identity of the chromophore that mediates blue light–induced damage should therefore be reexamined. Further, it is possible from experiments such as these to calculate the numbers of photons absorbed by rhodopsin and by the photoreversible intermediate(s). The calculations indicate that previously unsuspected large numbers of photon absorptions are occurring in blue light, and this insight could be valuable in understanding blue light–induced retinal damage.
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Footnotes |
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Supported by the Ernst and Berta Grimmke Foundation, Düsseldorf, Germany; the Schweizerische Unfallverhütungsanstalt Research Foundation, Luzern, Switzerland; the Bruppacher Foundation, Zurich, Switzerland; and Paolo Baiocchi, Intercast Europe, Parma, Italy. TPW received an Alexander Humboldt Scientist Award sponsored by R. Paulsen.
Submitted for publication November 4, 1999; revised March 1 and June 7, 2000; accepted July 5, 2000.
Commercial relationships policy: N.
Corresponding author: Christian Grimm, Department of Ophthalmology, University Eye Clinic, Frauenklinikstrasse 24, CH-8091 Zurich, Switzerland. cgrimm{at}opht.unizh.ch
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
; 550 nm; 409 µW/cm2) and of bleaching in
blue light (•; 403 nm; 300 µW/cm2) in the presence of
NH2OH were straight over several half-lives, indicating
that very little or no photoreversal of bleaching occurred in green
light or in blue light with NH2OH present. In contrast, the
line of rhodopsin bleaching in blue light (
; 403 nm; 300
µW/cm2) without NH2OH present was curvy,
indicating that bleaching in this condition is not a simple,
first-order process. The rate constants for rhodopsin bleaching in vivo
were determined from the slopes of the straight lines. The values are
Kb = 0.61 per minute (bleaching with
blue light in the presence of NH2OH) and
Kg = 2.1 per minute (bleaching with
green light).
; blue; 300 µW/cm2) after 5 minutes of green
light exposure and irradiation continued for an additional 1 minute.
Rhodopsin bleaching in green light generated photoproducts from which
rhodopsin (28% ± 15%) could be photoreversed by the additional blue
light exposure. Data points for green light bleach are from 
