From the Departments of Molecular Biology and
Genetics, ¶ Neuroscience, and
Ophthalmology and the
§ Howard Hughes Medical Institute, Johns Hopkins University
School of Medicine, Baltimore, Maryland 21205
Received for publication, November 7, 2000, and in revised form, January 5, 2001
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ABSTRACT |
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A large body of experimental and clinical data
have documented the damaging effects of light exposure on photoreceptor
cells although the identities of the biologically relevant molecular targets of photodamage are still uncertain. Several lines of evidence point to retinoids or retinoid derivatives as chromophores that can
mediate light damage. We report here that ABCR, a
photoreceptor-specific transporter involved in the recycling of
all-trans-retinal, is unusually sensitive to photooxidation
damage mediated by all-trans-retinal in vitro.
Partial loss of ABCR function is responsible for Stargardt macular
dystrophy, which is associated with accumulation of A2E, a diretinoid
adduct within the retinal pigment epithelium. Photodamage to ABCR
causes it to aggregate in SDS gels and results in the loss of
retinal-stimulated ATPase activity. Peripherin/RDS and ROM-1, two
structural proteins that colocalize with ABCR at the outer
segment disc rim, are also significantly more susceptible to
all-trans-retinal-mediated photodamage than are the major
proteins from the rod outer segment. These observations imply that
there may be specific protein targets of photodamage within the outer segment, and they may be especially relevant to assessing the risk of
light exposure in those individuals who already have diminished ABCR
activity due to mutation in one or both copies of the ABCR gene.
The retina is the only part of the central nervous system that is
directly exposed to light, and it has long been known that excessive
light exposure leads to retinal damage (1). Acute light toxicity, for
example that incurred by directly viewing a solar eclipse, has been
recognized since antiquity; in Plato's Phaedo, Socrates
advises that such injuries can be avoided by viewing the eclipse via
its reflection in water (2). Indirect exposure to sunlight can also
impair visual function if it occurs over an extended period. Among
military personnel exposed to bright sunlight and its reflection from
water or sand, temporary elevations in the threshold for dark
adaptation have been reported in individuals exposed without sunglasses
for 3-4 h each day over a 2-week period (3), and decreased visual
acuity and macular pigmentary changes have been reported following
several months of exposure (4-8). Of potentially greater significance
to public health is the effect that decades of exposure to `normal'
light levels may have on the retina and underlying retinal pigment
epithelium (RPE)1 (9). The
clinical relevance of this exposure is suggested by two recent
epidemiological studies showing a positive correlation between light
exposure and age-related macular degeneration, the most common
cause of visual impairment in the elderly (10-12).
An extensive body of work has documented the damaging effects of acute
and chronic light exposure in laboratory animals (reviewed in Refs.
13-15). Most of these studies have assessed the appearance of the
retina and RPE through the ophthalmoscope and in tissue sections using
light or electron microscopy; some studies have also assessed retinal
function via the electroretinogram. These studies have employed a
variety of animal species and light exposure protocols and, as a
result, have produced a number of disparate conclusions regarding the
initial cellular targets for light damage, the sensitivity of those
targets to different wavelengths and intensities, and the natural
history of tissue injury and repair. Despite the lack of a detailed
consensus, several broad themes have emerged, and these are summarized below.
Acute light damage involves both the photoreceptors and RPE, but the
relative susceptibilities of each cell layer and the subcellular
compartments within which damage occurs vary with wavelength (13-15).
Experiments in which rodents or monkeys are exposed to intense light
over a period of minutes to hours, as well as clinical data from humans
exposed to intense lights as an occupational hazard (e.g.
arc welders), indicate that light at the blue end of the visible
spectrum is ~50-fold more potent in producing acute damage than is
light from the midspectral region (2, 16-19). By contrast, chronic
exposure to relatively low light levels over a period of weeks or
months produces photoreceptor damage with an action spectrum that peaks
at ~500 nm and coincides with the absorption spectrum of rhodopsin
(20-22), strongly suggesting that visual pigment activation and/or
release of the all-trans-retinal chromophore plays a role in
light damage. A role for retinal or other retinoids in light damage is
further supported by the observations that (a) photoexcited
all-trans-retinal can generate singlet oxygen (23, 24) and
mediate photooxidative damage of lipids (25, 26), proteins (26, 27), or
DNA (28) in vitro (reviewed in Ref. 29), (b)
dietary vitamin A depletion partially protects the retina from chronic
light damage (30, 31), and (c) the retinas of RPE65( Although the identities of the biologically significant molecular
targets of photodamage are not yet known, the mechanisms by which they
are damaged have been inferred from experiments in which damage to the
retina is abrogated when an antioxidant or free radical scavenger is
administered prior to light exposure (33, 34). These experiments
indicate that photooxidative mechanisms play a significant role in the
generation of light damage. Consistent with this idea, a number of
investigators have documented photooxidation of lipids in photoreceptor
outer segments (OSs) following irradiation in vivo or
in vitro (25, 35, 36). By contrast, only a few studies have
addressed the question of protein targets of light damage within the
photoreceptor. These studies have demonstrated oxidation of the
rhodopsin sulfhydryl groups following irradiation of rod outer segments
(ROSs) in vitro (26, 27) and an ~2-fold decline in ROS
retinol dehydrogenase (RDH) activity 20 h after exposure of rats
to intense visible light (37).
One or more chromophores distinct from those involved in photoreceptor
damage are presumed to be responsible for light-mediated RPE damage. In
searching for the relevant chromophore(s) a number of investigators
have focused on the large quantities of age-dependent pigments, lipofuscin, that accumulate within the RPE (38-41). One major component of human RPE lipofuscin is a diretinal adduct, A2E,
that appears to form within the photoreceptor OS from the spontaneous
condensation of phosphatidylethanolamine and the
all-trans-retinal released from photoactivated rhodopsin
(42-44). Phagocytosis of OSs by the RPE results in accumulation of A2E
within the RPE where it appears to be trapped within phagolysosomes.
A2E efficiently absorbs blue light and is phototoxic to RPE cells in
culture (45); its progressive accumulation within the RPE suggests that
with age the RPE may become increasingly susceptible to phototoxic damage. Thus both photoreceptor and RPE photodamage may be mediated, at
least in part, by retinoids or retinoid derivatives.
The most recent line of evidence suggestive of a role for retinoids in
photodamage comes from the study of autosomal recessive Stargardt
disease. In Stargardt disease the cycling of retinoids between
photoreceptors and RPE, a process referred to as the visual cycle, is
defective. Stargardt disease arises from a partial loss of function of
ABCR (46-51), an ABC transporter that resides in the internal (disc)
membranes of photoreceptor OSs (52-54). Stargardt disease is the most
common form of early onset macular degeneration, and it is
characterized by a progressive accumulation of large quantities of
lipofuscin including A2E within the RPE, delayed dark adaptation, and a
progressive loss of central vision (43, 55-61). ABCR
gene mutations leading to partial loss of function can also cause
recessive cone-rod dystrophy (62), complete loss of ABCR function
causes autosomal recessive retinitis pigmentosa (62-64), and
heterozygosity for either of two sequence variants in the
ABCR gene has been implicated as a risk factor for
age-related macular degeneration (65). Purified and reconstituted ABCR
exhibits all-trans-retinal-stimulated ATP hydrolysis
in vitro (51, 66, 67), and ABCR knockout
mice exhibit an acute light-dependent accumulation of
all-trans-retinal within the OS and a progressive light-dependent accumulation of A2E in the RPE (43, 68).
Based on these data, ABCR appears to function as an
all-trans-retinal transporter/flippase that delivers
all-trans-retinal to RDH, the OS enzyme responsible for
conversion of released all-trans-retinal to
all-trans-retinol prior to its delivery to the RPE.
In this paper, we report that in vitro ABCR is unusually
sensitive to photooxidative damage mediated by
all-trans-retinal. If this type of photodamage also occurs
in vivo it would diminish the rate of reduction of
all-trans-retinal in the OS, and the resulting increase in
all-trans-retinal would be predicted to lead to further
phototoxic damage and an accelerated accumulation of A2E. Photodamage
of this type may be particularly relevant to understanding
pathophysiological mechanisms and assessing the risk of light exposure
in those individuals with inherited retinal diseases caused by partial
loss of ABCR function.
ROS Preparation--
Dark-adapted bovine ROSs were prepared by
the method of Papermaster (69). Unless otherwise noted, all ROS
preparations were used at a concentration of 1 mg/ml rhodopsin. For
depletion of both free and opsin-bound retinal, ROSs were exposed to
visible light for 60 min at room temperature in the presence of 10 mM hydroxylamine. An equal volume of BSA buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 4% BSA) was
added to the irradiated ROS suspension, and the ROSs were incubated on
ice for 20 min and recovered by centrifugation at 15,000 × g for 5 min at 4 °C. The pelleted ROSs were resuspended
and subjected to a second incubation in BSA buffer, recovered by
centrifugation, and stored in aliquots at SDS-Polyacrylamide Gel Electrophoresis and Immunoblot
Analysis of Recombinant ABCR, ROS ABCR, and Other ROS
Proteins--
Human ABCR was produced in transiently transfected 293 cells and azido-[32P]ATP labeling was performed with 302 nm irradiation from a Spectrolyne TR-302 transilluminator as described
in Ref. 51. For immunoblot assays of light damage, 293 membranes
containing ABCR or dark-adapted bovine ROSs in resuspension buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, and 3 mM MgCl2) were added to a 0.3-ml glass vial or
a 1.5-ml microcentrifuge tube, and either all-trans-retinal
(>95% pure by high pressure liquid chromatography; Sigma) in ethanol
or ethanol alone was added to a final ethanol concentration of 1%. In
a typical irradiation experiment, each 20 µl of reaction contained
0.5 mg/ml rhodopsin for ROS samples or 1 mg/ml total protein for 293 membrane samples as determined by the Bradford assay (BioRad). For UV
damage, samples in glass vials or uncapped microcentrifuge tubes were placed 5 cm from a longwave UV lamp (peak emission 366 nm; irradiance 2 mW/cm2; model UVL-56, UVP, Inc.) and irradiated from above
for the indicated length of time. For exposure to visible light,
samples in uncapped microcentrifuge tubes were placed in a room
temperature water bath and irradiated at a distance of 2 cm for 2 h from above with a fluorescent light source (F15T8-CW cool white 15 watt lamp; irradiance 1.5 mW/cm2; General Electric)
filtered through a 420-nm cut-off filter (GG-420, 1 mm thick, Schott
Glass Technologies, Inc.). Light sources were calibrated with a United
Detector Technologies model 61 optometer. After irradiation, an equal
volume of 2× SDS loading buffer was added, and the samples were
immediately loaded onto a 6% (see Figs. 3, 5, upper panel,
and 6) or 10% (see Figs. 1, 4, and 5, lower
panel) SDS-polyacrylamide gel electrophoresis resolving gel with a
5% stacking gel without sample heating. The gels were transferred to
nitrocellulose at 140 volts for 1 h at 4 °C. ABCR was detected
by immunoblotting using a rabbit polyclonal antibody against human ABCR
(ABCR1156-1258; Ref. 53), peripherin/RDS was detected with monoclonal
antibody 2B6, ROM-1 was detected by monoclonal antibody 1D5, the ATPase Assays of Photodamaged ABCR--
ROS ABCR was
purified, reconstituted into membranes, and assayed for ATPase as
described (51).
ROS GTPase Assays--
Each complete GTPase reaction contained 5 µl of ROSs and 100 µl of 1× GTPase buffer (20 mM
Hepes, pH 7.5, 50 mM KCl, 5 mM MgCl2, 100 µM GTP containing 106 cpm
[ Rhodopsin Reconstitution--
10 mM
all-trans-retinal in ethanol was added to retinoid-depleted
ROSs in 20 mM Hepes, pH 7.5, 50 mM KCl to give
a final all-trans-retinal concentration of 100 µM. Control ROSs received only ethanol. Following incubation in the dark or exposure to 366 nm light for 10 min as
described for ABCR assays, 30 µl of aliquots of the ROS samples were
added to 400 µl of 20 mM Hepes, pH 7.5, 50 mM
KCl, 5 mM MgCl2, 10% glycerol, 0.05% BSA,
0.6% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
(CHAPS), and 50 µM 11-cis-retinal, and the
mixtures were incubated at room temperature in the dark for 10 min.
Preliminary experiments demonstrate that under these conditions
reconstitution with 11-cis-retinal proceeds with a half-life
of less than 1 min. Hydroxylamine was then added to a final
concentration of 50 mM, and the incubation continued for an
additional 10 min at which point the samples were frozen on dry ice.
Photobleaching difference spectra were obtained in a water-jacketed
cuvette by bleaching with an intense visible light delivered to the
cuvette by a fiber optic cable. All spectra were recorded with a Uvikon
860 spectrophotometer and analyzed using Cricket Graph software.
RDH Assays--
Recombinant bovine RDH was prepared by transient
transfection of 293 cells with a RDH expression plasmid followed by
membrane purification as described in ref. 70. ROS RDH activity was
measured starting with retinoid-depleted ROSs.
All-trans-retinal addition to a final concentration of 100 µM and irradiation with 366 nm light for 10 min were
performed with the 293 and ROS membranes as described above for the ROS
GTPase assays. The RDH reaction was initiated by solubilizing the 293 or ROS membranes, which had been stored on dry ice following the
pretreatment regimen in 400 µl of 20 mM Hepes, pH 7.5, 50 mM KCl, 5 mM MgCl2, 10% glycerol, 0.05% BSA, 0.6% CHAPS, 100 µM NADPH, and 50 µM all-trans-retinal. The reaction was
monitored spectrophotometrically as described in Ref. 70. A control
reaction containing 100 µM NADH in place of NADPH showed
barely detectable RDH activity.
Protein Targets of All-trans-retinal-mediated Photodamage in
Vitro--
The investigation reported here began with an experiment to
determine whether all-trans-retinal affects the affinity of
ABCR for ATP. To this end we asked whether all-trans-retinal
alters the efficiency of photo-cross-linking of
azido-[
These observations suggested the possibility that
all-trans-retinal-dependent photodamage might
impair ABCR function in the intact human eye. In humans, the lens
protects the retina from UV damage by efficiently absorbing light of
<~410 nm (Fig. 2; Ref. 71). Although
the peak absorption of all-trans-retinal is at 380 nm, the
longwave limb of the absorption curve produces appreciable absorption
at wavelengths >410 nm as seen in Fig. 2, suggesting that ambient
light might produce a significant level of photoexcitation of
all-trans-retinal within the human eye. We therefore asked
whether all-trans-retinal-dependent aggregation of ABCR can be produced by visible light filtered to approximate the
filtering effect of a human lens. As seen in Fig.
3, a 2-h exposure to visible light of
>410 nm produces aggregation of ABCR that increases with increasing
concentration of all-trans-retinal. The loss of monomeric
ABCR is more pronounced in 293 membranes than in ROS membranes, a
difference that may relate to differences in lipid composition or to
the high levels of antioxidants in ROS membranes, as discussed more
fully below. In many of the experiments reported below, we have used a
brief (10-20 min) exposure to 366 nm light rather than an extended
exposure to >410 nm light because the shorter wavelength light
produces efficient photoexcitation of all-trans-retinal and
aggregation of ABCR and its use minimizes the problem of competing
reactions that might occur during an extended incubation time.
We next asked whether other ROS proteins are also subject to light- and
all-trans-retinal-dependent aggregation. As seen
in Fig. 4, there is little or no
aggregation of two integral membrane proteins, the
As noted in the introduction, it has long been known that
photoexcitation of all-trans-retinal leads to the production
of singlet oxygen (23, 24). To determine whether ABCR and
peripherin/RDS aggregation is oxygen-dependent, increasing
concentrations of all-trans-retinal were added to ROSs in
room air, 100% oxygen, or argon and exposed to 366 nm light (Fig.
5). In this experiment, efficient
aggregation of both proteins was observed in room air and in 100%
oxygen but not in argon, strongly suggesting that all-trans-retinal-dependent photodamage involves
the production of oxygen radicals.
The experiments described above involve the addition of exogenous
all-trans-retinal to ROS or 293 membranes. In the
experiments with ROS membranes, the rhodopsin concentration in the
reaction mixture is 12.5 µM, and therefore 25 µM exogenous all-trans-retinal, the lowest
concentration tested, corresponds to twice the concentration of
rhodopsin-bound retinal. In vivo, free
all-trans-retinal is released from the photoactivated visual
pigment and is subsequently reduced within the OS to
all-trans-retinol by RDH in an NADPH-dependent reaction. Conversion of all-trans-retinal to
all-trans-retinol would be predicted to dramatically reduce
photooxidation by >410 nm light because the absorption spectrum of
all-trans-retinol is blue-shifted by ~55 nm relative to
that of all-trans-retinal (Fig. 2). In addition,
all-trans-retinol has been reported to have modest
antioxidant activity (72). To determine whether light-dependent aggregation of ABCR is promoted by
endogenous all-trans-retinal and whether conversion of
all-trans-retinal to all-trans-retinol protects
ABCR from aggregation, dark-adapted ROSs were exposed to >410 nm or
366 nm light in the presence or absence of NADPH without exogenous
all-trans-retinal (Fig. 6, left). This experiment reveals appreciable
light-dependent aggregation of ABCR that is largely
abrogated by the inclusion of NADPH during the period of irradiation.
The protective action of NADPH presumably reflects enzymatic conversion
of endogenous all-trans-retinal to
all-trans-retinol because no protection is seen upon
addition of NADH or NADP (Fig. 6, right), neither of which
can support reduction by photoreceptor RDH (70, 73).
Functional Assays of in Vitro Photodamage--
The aggregation of
ABCR observed in SDS gels presumably reflects a significant structural
alteration and suggests a corresponding functional impairment. In
vitro, ABCR exhibits ATPase activity, which is stimulated by
all-trans-retinal, a coupling that is presumed to reflect
the vectorial transport of all-trans-retinal driven by ATP
hydrolysis. At present, no in vitro transport assay exists for ABCR. In an initial experiment to assess the functional effects of
photodamage, we immunoaffinity purified ABCR, reconstituted it into
lipid membranes, and asked whether ATPase activity was affected by
exposure to 366 nm light in the presence of 20 µM all-trans-retinal. As seen in Fig.
7A, irradiation of ABCR in the
presence of 20 µM all-trans-retinal led to an
~60% loss of ATPase activity relative to a control reaction in which
all-trans-retinal was irradiated prior to the addition of
ABCR. As shown below, most of this loss of ATPase activity is referable
to that part of the total ATPase activity that is stimulated by
all-trans-retinal.
In a second experiment, irradiation of ABCR was carried out in the
presence of a variable concentration of all-trans-retinal to
determine whether there was a dose-dependent loss of basal or retinal-stimulated ATPase activity (Fig. 7B). Control
reactions in which various concentrations of
all-trans-retinal were irradiated prior to the addition of
ABCR show no loss of ATPase with increasing concentration of irradiated
all-trans-retinal (open squares); indeed, this
set of samples shows an increase in ATPase activity with increasing
concentrations of preirradiated all-trans-retinal indicating
that irradiated all-trans-retinal still retains its ability
to stimulate the ABCR ATPase activity, consistent with the previously
observed stimulatory activity of various retinal isomers (66). By
contrast, an otherwise identical set of samples in which ABCR and
variable concentrations of all-trans-retinal were irradiated
together show no stimulation of ATPase activity with increasing
concentrations of irradiated all-trans-retinal (open
circles). Addition of fresh all-trans-retinal to a
final concentration of 30 µM produces an ~2-fold
increase in ATPase activity over the basal level (filled
squares), and this increase is inhibited in a
dose-dependent manner by prior irradiation of ABCR in the
presence of all-trans-retinal (filled circles)
with nearly complete inhibition at 30 µM
all-trans-retinal. This experiment reveals that
all-trans-retinal-dependent photodamage has
little effect on the basal ATPase activity of ABCR (compare the data points represented by open circles and open
squares). As site-directed mutagenesis shows that most of the
basal ATPase activity appears to be derived from the first nucleotide
binding domain (51), the selective loss of retinal-stimulated ATPase
may reflect photodamage to the transmembrane transport domain and/or to
the second nucleotide binding domain with little or no damage to the
first nucleotide binding domain.
To extend the functional analysis of ABCR described above to other ROS
proteins, we examined the effect of irradiation in the presence or
absence of exogenous all-trans-retinal on the regeneration of opsin within ROS membranes, the activity of ROS GTPase, and the activity of ROS and recombinant RDH. For the first three assays we used ROSs that had been largely depleted of endogenous retinoids by extensive photobleaching in the presence of hydroxylamine followed by washing with BSA. As seen in Fig.
8, exposure of retinoid-depleted ROSs to
366 nm light in the presence of 100 µM
all-trans-retinal had no effect on the subsequent
regenerability of opsin with fresh all-trans-retinal. By
contrast, ROS GTPase activity was reduced ~40%, and ROS RDH activity
was reduced ~30% relative to unirradiated controls. Similar
treatment of recombinant RDH in 293 cell membranes produced an ~60%
loss in activity consistent with a protective effect of ROS membranes
relative to 293 membranes as noted above for ABCR photodamage. The
decrease in ROS GTPase and ROS RDH activities in samples exposed to UV
light without added all-trans-retinal relative to the
samples that were not exposed to UV light may reflect photodamage
mediated by residual endogenous all-trans-retinal. The
modest decreases in ROS RDH and GTPase activities following light
exposure in the presence of 100 µM
all-trans-retinal indicates that these proteins are
relatively resistant to photodamage.
The principal conclusions of this study are that irradiation of
ABCR in the presence of all-trans-retinal and oxygen
in vitro leads to its rapid modification and functional
inactivation. ABCR, together with peripherin and ROM-1, are
significantly more susceptible to all-trans-retinal-mediated
photodamage than are the major membrane and membrane-associated
proteins from transfected 293 cells or from ROSs. ROS RDH and GTPase
activities show moderate susceptibility to
all-trans-retinal-mediated photodamage, but the
regenerability of opsin is unaffected under the same conditions.
All-trans-retinal-mediated photodamage is produced
efficiently by longwave UV light, but it is also produced by light that
has been filtered to approximate the wavelength distribution impinging
on the human retina. ABCR photodamage occurs more readily in membranes
from 293 cells than from ROSs consistent with the previously described
high concentration of antioxidants in ROSs (72, 74). Following release
of endogenous all-trans-retinal from rhodopsin in
photobleached ROSs, ABCR photodamage can be significantly abrogated by
addition of NADPH to the preparation presumably by promoting the
reduction of all-trans-retinal to all-trans-retinol (Fig. 9).
Finally, ABCR photodamage selectively eliminates retinal-stimulated but
not basal ATPase activity, suggestive of damage to the transmembrane
transport domain. These data suggest the interesting possibility that
the susceptibility of ABCR to all-trans-retinal-mediated
photodamage is related to the presumed function of ABCR as a retinal
transporter.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
)
mice, which have little or no rhodopsin secondary to defective
production of 11-cis-retinal in the RPE, are highly
resistant to acute light toxicity (32).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C.
subunit of the cGMP channel was detected with monoclonal antibody 1D1
(gifts of Dr. R. Molday), and arrestin was detected with rabbit
polyclonal antisera (a gift of Dr. T. Shinohara). Following incubation
with a horse radish peroxidase-conjugated secondary antibody, the
immunoblots were developed using the SuperSignal West Pico
chemiluminescent substrate (Pierce).
-32P]GTP). Prior to addition of 1× GTPase buffer, 10 mM all-trans-retinal in ethanol was added to an
aliquot of dark-adapted ROSs to give a final
all-trans-retinal concentration of 100 µM. A
control aliquot of ROSs received only ethanol. The ROS samples were
then exposed to room light for 1 min, an exposure sufficient to bleach
>90% of the rhodopsin. Half of each sample was further exposed to 366 nm light as described for ABCR assays for 10 min at room temperature while the other half was kept in the dark. The GTPase reaction was
initiated by the addition of 1× GTPase buffer and incubated at
37 °C under dim red light. At 5 min intervals, 15 µl of aliquots were removed from each reaction and vigorously mixed with a 200-µl suspension of activated charcoal; released 32P was measured
in the supernatant after removing the charcoal by centrifugation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP to ABCR expressed in 293 cells.
As seen in Fig. 1A,
all-trans-retinal produces a dose-dependent loss
in 32P labeling of ABCR at the expected monomer mobility of
~250 kDa. By contrast, azido-[32P]ATP
photo-cross-linking to an endogenous protein of ~55 kDa is unaffected
by addition of all-trans-retinal. Although this result might
be interpreted as evidence for an inhibition by
all-trans-retinal of azido-ATP binding to ABCR,
immunoblotting with anti-ABCR antibodies revealed instead that exposure
of ABCR to UV light (the regimen used for azido-ATP
photo-cross-linking) in the presence of all-trans-retinal, converts ABCR from a species that migrates as an apparent monomer in
SDS gels to one that appears to be aggregated and is largely retained
at the origin (Fig. 1B). This all-trans-retinal-
and UV light-dependent aggregation of ABCR is independent
of azido-ATP. Moreover, a comparison of the bulk proteins within 293 membrane preparations by Coomassie Blue staining indicates that
aggregation is remarkably selective for ABCR (Fig. 1B).
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Fig. 1.
The loss of azido-[32P]ATP
labeling of ABCR produced by all-trans-retinal
reflects aggregation of ABCR. A,
azido-[32P]ATP labeling of 293 cell membranes from cells
expressing ABCR (left) or from nontransfected cells
(right). The samples were exposed to 302 nm light for 10 min
at room temperature in the presence of the indicated concentrations of
all-trans-retinal. A protein of ~55 kDa (small
arrowhead) is radiolabeled with azido-ATP in 293 membranes and is
unaffected by all-trans-retinal addition. ABCR (large
arrowhead) migrates at ~250 kDa. B, Coomassie Blue
stain of membrane proteins from transfected 293 cells (left)
and the corresponding immunoblot with anti-ABCR antibodies
(right) following a 10-min irradiation with 366 nm light in
the presence of the indicated concentrations of
all-trans-retinal. Bars at right indicate from
top to bottom the molecular mass standards of
220, 130, 90, 70, 60, 40, and 30 kDa.
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Fig. 2.
Absorption spectra of retinoids and
filters. Upper panel, all-trans-retinol
(ROH, absorption maximum 325 nm),
all-trans-retinal (RAL, absorption maximum 380 nm), rhodopsin (RHO, absorption maximum 500 nm). Lower
panel, the human lens (71) and a shortwave cutoff filter (1 mm
thick GG-420; Schott Glass Technologies, Inc.) used to approximate the
human lens for irradiation experiments with visible light. Both the
lens and the GG-420 filter exhibit an absorbance >1 at wavelengths
<410 nm (OD).
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Fig. 3.
Visible light and
all-trans-retinal induce dose-dependent
aggregation of both expressed ABCR and ROS ABCR in
vitro. Samples containing the indicated concentrations
of all-trans-retinal were irradiated for 2 h with a
fluorescent light filtered through a GG-420 shortwave cutoff filter.
For a given all-trans-retinal concentration, aggregation of
ABCR is greater in 293 membranes than in ROSs. Bars at
right indicate from top to bottom the
molecular mass standards of 220, 130, 90, and 70 kDa.
subunit of the
cGMP gated channel and rhodopsin, or of two peripheral membrane
proteins, arrestin and rhodopsin kinase. By contrast, peripherin/RDS
and ROM-1, homologous integral membrane proteins that, like ABCR, are
localized to the OS disc rim, aggregate upon exposure of ROSs to
all-trans-retinal and 366 nm light as does ABCR. At present,
the chemical basis for protein aggregation is unknown. The resistance
of the aggregates to incubation in 2% SDS and 5%
-mercaptoethanol
at room temperature suggests that aggregation involves covalent
cross-links distinct from disulfide bonds.
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Fig. 4.
UV light and
all-trans-retinal induce dose-dependent
aggregation of a subset of ROS membrane proteins in
vitro. Irradiation was with a 366-nm light for 10 min.
Both the stacking and resolving gels are shown. Left,
Coomassie Blue stain of ROS proteins. Large arrowhead
indicates the position of monomeric ABCR; small arrowhead
indicates opsin. Right, immunoblotting results for six
ROS proteins. ABCR, peripherin/RDS, and ROM-1 show
all-trans-retinal-dependent aggregation;
arrestin, the subunit of the cGMP-gated channel, rhodopsin kinase,
and the major Coomassie Blue-stained proteins do not. Bars
at right indicate from top to bottom
the molecular mass standards of 220, 130, 90, 70, 60, 40, and 30 kDa.
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Fig. 5.
UV light and
all-trans-retinal promote aggregation of ABCR and
peripherin/RDS in vitro in an
oxygen-dependent reaction. Samples of ROSs containing
the indicated concentrations of all-trans-retinal were
sealed in glass vials, and the atmosphere within the vial was either
maintained as room air, exchanged for argon, or exchanged for oxygen
prior to irradiation for 10 min with 366 nm UV light. The
upper immunoblot probed with anti-ABCR antibodies shows both
stacking and resolving gels; the lower immunoblot probed
with anti-peripherin/RDS antibodies shows only the resolving gel.
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Fig. 6.
NADPH but not NADH protects ROS ABCR from UV
and visible light damage in vitro in the absence of
exogenous all-trans-retinal. Wavelengths and
times of irradiation are indicated. NADP, NADH, or NADPH was added to a
final concentration of 1 mM to the indicated tubes. Of the
three compounds only NADPH confers protection, presumably reflecting
the reduction of released all-trans-retinal by ROS RDH.
Bars at right indicate the molecular mass
standards as described for Fig. 3.
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Fig. 7.
UV light and
all-trans-retinal produce a dose-dependent
loss in the ATPase activity of purified and reconstituted ABCR.
The schematic diagram at the bottom shows (from
left to right) the order of addition of
components and of irradiation for each of the sample sets. Irradiation
was at 366 nm for 10 min in all cases. A,
all-trans-retinal at 20 µM was irradiated
either in the presence or absence of reconstituted ABCR, and then ABCR
ATPase was monitored in the presence of fresh
all-trans-retinal added to the indicated final
concentrations. Note that for panel A 100% ATPase activity
refers to the activity level in the presence of 20 µM
preirradiated all-trans-retinal. B, the indicated
concentrations of all-trans-retinal were irradiated either
in the presence or absence of ABCR, and then ABCR ATPase was monitored
in the presence or absence of 30 µM fresh
all-trans-retinal. The final concentrations of lipid and
ABCR were identical in all samples. Note that for panel
B 100% ATPase activity refers to the basal activity level,
i.e. the activity in the absence of added
all-trans-retinal.
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Fig. 8.
Effect of all-trans-retinal
and UV light exposure in vitro on reconstitution of
bovine opsin with 11-cis-retinal (A),
ROS GTPase activity (B), ROS RDH activity
(C), and recombinant RDH activity in transfected 293 membranes (D). Samples were exposed to the
following pretreatments: a 10-min incubation in the dark (-light,
-retinal; open circles in B-D), irradiation
with 366 nm light for 10 min (+light, -retinal; filled
circles in B-D), irradiation with 366 nm light for 10 min in the presence of 100 µM
all-trans-retinal (+light, +retinal; filled
squares in B-D). ROSs were largely depleted of
endogenous retinoid prior to UV irradiation as described under
"Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Schematic diagrams showing the visual cycle
and the characteristics that are known or are predicted to change along
the length of the ROS. A, the principal transformations
of the visual cycle that occur within the ROS. Three aberrant fates for
released all-trans-retinal are shown at the top
of the diagram. The absorption maxima of the relevant retinoids are
indicated. The surface membranes of the ROS and RPE are indicated by
horizontal lines. B, a rod cell (left)
and summary plots (right) indicating, as a function of
distance along the outer segment, the age of the discs (for mammals),
oxygen tension (data from cats and monkeys; Refs. 88, 89), recovery
time following an adapting stimulus (data from toads; Ref. 75), and the
predicted accumulation of photodamage as described under
"Discussion."
All of the experiments described here were performed in vitro. It will therefore be important to determine the extent to which this type of photodamage occurs in the living eye and in which species and under which conditions it occurs. In the paragraphs that follow, we relate the present observations to the cell biology and physiology of photoreceptors and to the pathophysiology of degenerative retinal disease under the working hypothesis that photodamage of the type described here can occur in the living human eye.
ABCR, All-trans-retinal, and the Gradient of Recovery of Photosensitivity along the OS-- Photoreceptor OS renewal occurs at a constant rate throughout life. New ROS constituents are synthesized within the photoreceptor cell body and as new discs are added at the base of the ROS older discs are progressively displaced to more distal locations within the ROS until they are engulfed and degraded by the RPE. Because discs move along the ROS without exchanging their integral membrane protein components, these proteins age as a cohort. In mammals, 10% of the ROS length is turned over per day, and therefore the membrane proteins at the distal end of each ROS are uniformly 10 days old (Fig. 9B).
It has long been assumed that ROS turnover exists as a mechanism for eliminating proteins or lipids that are impaired because of accumulated damage (9). Evidence that discs exhibit a progressive functional impairment as they age comes from suction electrode recordings in which different regions along the OS of a toad rod are exposed to an adapting light. After the period of adaptation, the time course of local recovery of sensitivity is measured with a series of dim test lights (75). In dark-adapted rods, test flashes elicit responses of nearly identical amplitude independent of position along the OS, indicating little or no effect of disc age on the efficiency of the phototransduction cascade. However, following a stimulus estimated to bleach several percentage of the rhodopsin within the irradiated OS region, there is a delay in recovery of sensitivity that increases with increasing distance from the inner segment (Fig. 9B). Additional experiments in which an adapting stimulus delivered to the proximal OS was found to have no effect on the recovery kinetics of the distal OS suggest that the delay in recovery of the distal OS does not reflect limiting amounts of a diffusable substance from the inner segment but rather is an intrinsic property of the distal OS.
A number of studies have pointed to opsin, released all-trans-retinal, and/or other as yet unidentified products of photobleaching as potential sources of transduction noise, which limit photosensitivity during recovery from an adapting stimulus (Fig. 9A). For example, binding of all-trans-retinal to opsin in vitro leads to a low level of activation (76-78), and in vivo the initial rise and subsequent decline in all-trans-retinal following intense illumination parallels the loss and recovery of visual sensitivity (79-84). These studies suggest that reduction of all-trans-retinal to all-trans-retinol by the combined action of ABCR and RDH plays a role in recovery of photosensitivity by lowering the concentration of free all-trans-retinal. In keeping with this idea, Stargardt disease patients and ABCR knockout mice exhibit a pronounced delay in dark adaptation (58, 68). With respect to the delay in the recovery of photosensitivity in the distal OS described in the preceding paragraph, a decrement in ABCR activity secondary to accumulated photodamage would be predicted to slow the rate of reduction of all-trans-retinal and could therefore contribute to the observed delay in recovery of sensitivity.
Following a strong bleaching light, direct measurements of the rate of reduction of all-trans-retinal along the length of the OS, made by observing the fluorescence of all-trans-retinol, show a spatially uniform accumulation of all-trans-retinol during the ensuing 10-20 min (85). However, extrapolation of these data to physiological bleaching levels is difficult because the experimental requirement for extensive photobleaching releases all-trans-retinal in quantities far above saturation for RDH, and this may mask local differences in the efficiency of ABCR-mediated transport. Whether reduction of all-trans-retinal occurs more slowly in the distal OS following an adapting light in the physiological intensity range remains an open question.
All-trans-retinal and the Gradient of Photodamage along the OS-- In both rats and monkeys, acute exposure to broad spectrum visible light produces a disorganization of photoreceptor OSs that is most prominent in the distal region of the OS where the discs are oldest (34, 86, 87). Assuming that photooxidative damage via all-trans-retinal represents one of the mechanisms of this damage, we suggest that this spatial pattern could reflect the following several factors either singly or in combination: (a) an intrinsic vulnerability of older discs perhaps related to the cumulative effect of previous photodamage; (b) the proximity to the choroidal blood supply, which produces an oxygen concentration gradient such that the distal OS is at a pO2 of ~100 mm of mercury (equivalent to arterial blood), whereas the proximal OS is at a pO2 of ~10 mm of mercury in the dark and ~30 mm of mercury in the light as a result of its proximity to the mitochondria-rich inner segment, the principal region of oxygen consumption within the retina (Fig. 9B, Refs. 88 and 89); (c) the proximity to the RPE, which in the mammalian retina, produces more rapid rhodopsin regeneration in the distal OS (90) presumably because of more efficient access to RPE stores of 11-cis-retinal; and (d) the greater distance from the inner segment, which could limit access to NADPH or substrates coupled to the local production of NADPH by ROS glucose-6-phosphate dehydrogenase (91, 92) thereby limiting the rate of reduction of all-trans-retinal to all-trans-retinol. We speculate that the morphological disorganization of OS discs following light damage might reflect all-trans-retinal-mediated photooxidative damage to peripherin/RDS and/or ROM-1, which together are proposed to form part of the scaffold that maintains discs morphology (93) and which, as shown here, are both highly susceptible to all-trans-retinal-mediated photodamage.
The Visual Cycle, All-trans-retinal-mediated Photodamage, and A2E-- All-trans-retinoids released by photobleaching of rhodopsin flow from the OS to the RPE to be reisomerized and then returned to the OS (Fig. 9A). Reduction of all-trans-retinal to all-trans-retinol appears to be the rate-limiting step in the visual cycle as measured in the living rodent eye (81, 84). As noted in the introduction, a small fraction of all-trans-retinal, together with OS phosphatidylethanolamine, reacts to form A2E. This reaction requires the condensation of two all-trans-retinal molecules, and therefore the rate of A2E production should be proportional to the square of the concentration of free all-trans-retinal. Because a brief high intensity light stimulus will lead to a high peak concentration of free all-trans-retinal, such a stimulus will lead to the formation of more A2E than would the same number of photons delivered as a prolonged low intensity stimulus. Moreover, those stimuli that are strong enough to release quantities of all-trans-retinal that saturate the ABCR/RDH pathway will lead to the accumulation of free all-trans-retinal for an extended period within the OS.
Complete loss of ABCR gene function in mice leads to
an accumulation of all-trans-retinal within the OS following
a light stimulus, and complete or partial loss of
ABCR gene function in mice or humans leads to an
increase in the accumulation of A2E in the RPE (43, 68). Once formed,
A2E is extremely stable as judged by the observation that placing
ABCR(/
) mice in the dark does not detectably
lower the level of A2E that has already accumulated within the RPE
(43). If this result can be extrapolated to humans and to time scales
of decades rather than months, it would suggest that small decrements
in ABCR function may be of clinical significance because small
increases in A2E production would be cumulative. By contrast,
decrements in the function of phototransduction or structural proteins
would not be expected to produce a cumulative effect. Based on the
logic of the preceding paragraph, it is reasonable to suppose that
inherited ABCR defects also lead to an increase in
all-trans-retinal-mediated photooxidation products within
the OS, which, like A2E, could accumulate within the RPE. Presumably
photodamage to ABCR would lead to the same constellation of effects
observed with inherited ABCR defects.
Photodamage and OS Degradation within the RPE-- Each human RPE cell contacts ~50 rod OSs, and therefore each RPE cell engulfs and degrades the equivalent of five OSs/day (94), a level of constitutive phagocytic activity greater than that of any other cell in the human body. As noted above, one obvious risk in such a system is that relatively small quantities of nondegradable compounds produced within the OS may, over time, accumulate to appreciable levels within the RPE, a problem compounded by the minimal turnover of RPE cells in vivo (95). The problem of nondegradable compounds may be especially acute with photooxidation products, which are likely to encompass a large variety of molecular structures, at least some of which may not be substrates for lysosomal enzymes, and which therefore may accumulate in lysosomes/phagosomes. By comparison, keratinocytes, the principal class of light-exposed cells in the skin, are shed externally so that there is no requirement for lysosomal processing of photooxidation products. These considerations emphasize the advantages of minimizing photooxidative damage within the OS especially in species that are long-lived.
Protective Mechanisms within the OS-- The significance of photooxidation for the retina is underscored by the long-standing observation that photoreceptor OSs are rich in endogenous antioxidants, including vitamin E, glutathione, and taurine (71, 73). The greater susceptibility of ABCR to photooxidative damage when irradiated in 293 versus ROS membranes (Fig. 3) demonstrates the functional protection afforded by these ROS anti-oxidants. A second protective strategy within the OS is the reduction of all-trans-retinal to all-trans-retinol (Fig. 9A). In addition to decreasing any effects that all-trans-retinal may have in generating postbleaching noise, reduction of all-trans-retinal would be expected to decrease photodamage because the all-trans-retinol absorption spectrum is blue shifted ~55 nm relative to that of all-trans-retinal (Fig. 2). In humans, the ~410-nm shortwave cutoff provided by the lens effectively eliminates light absorption by all-trans-retinol but admits light that can be absorbed by the longwave tail of the all-trans-retinal absorption spectrum (Fig. 2). Moreover, in vivo, the protective effect of reduction of all-trans-retinal may be even greater than the comparison between these two absorption spectra might indicate because all-trans-retinal in the OS efficiently forms Schiff bases with phosphatidylethanolamine. As the pKa of these Schiff bases is ~6 (96), 5-10% of them will be protonated at physiological pH, and these will absorb maximally at 440 nm. Finally, sequestration of free retinal by intracellular retinoid-binding proteins would be expected to protect other outer segment constituents from photodamage.
Implications for Retinal Disease Susceptibility--
If
accumulation of A2E and/or OS-derived photooxidation products represent
a risk factor for retinal disease then by implication light exposure
per se should also constitute a risk factor both because
light promotes the release of all-trans-retinal from
bleached visual pigment and because light absorption by released
all-trans-retinal promotes photooxidation (Fig.
9A). The efficiency with which all-trans-retinal produces ABCR photodamage in vitro suggests an additional
risk of light exposure: partial inactivation of ABCR with a resulting decrease in the rate of reduction of all-trans-retinal. The
observation that partial loss of ABCR function by gene mutation
produces progressive retinal disease is consistent with the hypothesis
that any environmental processes that lead to a decrease in ABCR
function may increase the risk or accelerate the development of retinal
disease. An environmental effect of this type may be most relevant to
individuals with partial loss of ABCR activity due to gene mutation(s)
because a decrease in ABCR activity would presumably have a greater
adverse impact in this context than it would in the context of normal ABCR function.
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ACKNOWLEDGEMENTS |
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We thank Drs. R. Molday and T. Shinohara for antibodies; Dr. A. Rattner for recombinant RDH; Drs. K.-W. Yau and V. Kefalov for helpful discussions; and N. Thekdi, Dr. A. Rattner, and two anonymous reviewers for helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported by the Howard Hughes Medical Institute and the National Eye Institute National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: 805 Preclinical Teaching Building, 725 N. Wolfe St., Johns Hopkins Univ. School of Medicine, Baltimore, MD 21205. Tel.: 410-955-4679; Fax: 410-614-0827; E-mail: jnathans@jhmi.edu.
Published, JBC Papers in Press, January 23, 2001, DOI 10.1074/jbc.M010152200
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ABBREVIATIONS |
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The abbreviations used are: RPE, retinal pigment epithelium; OS(s), outer segment(s); ROS(s), rod outer segment(s); RDH, retinol dehydrogenase; ABC, ATP-binding cassette; BSA, bovine serum albumin; mW, milliwatt.
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