Retinoid cycles in the cone-dominated chicken retina
Department of Biology, the University of Texas at San Antonio, San Antonio, TX 78249, USA
* Author for correspondence (e-mail: atsin{at}utsa.edu)
Accepted 13 September 2005
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Summary |
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Key words: cone, visual cycle, retinyl ester, vitamin A
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Introduction |
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In humans, daylight vision is primarily mediated by cone photoreceptors.
Cone chromophore regeneration is several fold faster than rod chromophore
regeneration in light conditions (Perry
and McNaughton, 1991). Cone, but not rod, visual pigment from
isolated frog retina can spontaneously regenerate
(Goldstein, 1967
;
Hood and Hock, 1973
). Mata et
al. (2002
) have shown that
membrane fractions prepared from cone-dominated retina contain three key
visual cycle enzymatic activities, including those of retinol isomerase,
11-cis retinyl ester synthase and 11-cis retinol
dehydrogenase. Data from our laboratory and others have shown that the retinae
of cone-dominated species such as chicken and ground squirrel store a larger
amount of retinyl ester than those in the RPE, of which the 11-cis
isomer is more abundant (Rodriguez and
Tsin, 1989
; Das et al.,
1992
; Mata et al.,
2002
). These experiments suggest that at least some vitamin A
chromophore for cone pigment (iodopsin) may come from a location within the
retina and may follow a separate visual cycle pathway
(Jin et al., 1994
;
Bustamante et al., 1995
).
In the present study, to further the understanding of this proposed cone visual cycle, we quantitatively characterize the change in retinoids in chicken retina and RPE during light and dark adaptation. We show that light exposure increases 11-cis retinyl esters in the chicken retina and all-trans retinyl esters in the RPE. Upon dark adaptation, the 11-cis retinyl ester pool in the retina declines at a rate about nine times faster than that of the all-trans retinyl pool in the RPE. Furthermore, isolated retinae devoid of RPE in vitro show similar light-initiated accumulation of 11-cis retinyl esters in concert with a depletion of 11-cis retinal, confirming the RPE independence of the accumulation of this 11-cis retinyl ester pool. Finally, to begin the characterization of the kinetics of such a cone visual cycle, we have collected experimental evidence to show a light-dependent accumulation/depletion cycle of 11-cis retinyl esters in the chicken retina during visual pigment bleaching and regeneration. Based on the amount of 11-cis retinyl esters and its fast depletion rate, we conclude that this 11-cis retinyl ester pool is the primary source of the visual chromophore for cone pigment regeneration.
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Materials and methods |
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Change in retinoids in isolated retina
Retinae were dissected (under dim red light) from dark-adapted (4 h) adult
chicken eyes (from Tyson Foods, Inc.). Tissue was placed in Tyrode's avian
buffer and exposed to different durations of light exposure (bleached 80%
visual pigments; 2500 lux from two 90 W halogen bulbs at 30 cm from
sample) in vitro. Tissues were homogenized in Tyrode's avian buffer
under dim red light.
Characterization of retinoid cycle in young chicken
Young chickens (60 g) were purchased from Producer's Coop (Seguin,
Texas, USA) and dark adapted overnight before being exposed to two 90 W
halogen bulbs (bleached 70% visual pigments; about 2000 lux) for the indicated
durations. Chicken were anesthetized by CO2, decapitated, and,
after eye removal, retina was dissected free of RPE. Tissues were homogenized
in Tyrode's avian buffer under dim red light.
Extraction and HPLC analyses of retinoids
Ocular tissues were homogenized in 10 mmol l-1 Tris-HCl (pH
7.5), and retinoids were extracted by the addition of ethanol and hexane.
Retinyl esters were analyzed by gradient HPLC (0.2 to 10% dioxane in hexane, 2
ml min-1; 4.6x250 cm, 5 µm silica column) according to the
method described by Mata et al.
(2002; see retinyl esters in
Fig. 1). Gradient HPLC results
indicated no detectable amounts of retinol in light- or in dark-adapted
chicken retina or RPE. Therefore, hexane extracts prepared from retina and RPE
samples in subsequent experiments were first saponified in strong base (0.33
mol l-1 KOH in ethanol), and the retinol products (derived from
retinyl ester) were analyzed by isocratic HPLC (717plus Waters autosampler,
Waters 515 pump with a photodiode array detector; Milford, MA, USA) with 10%
dioxane/hexane eluting through a 4.6x250 cm, 5 µm silica column at 2
ml min-1 (i.e. retinyl esters in Figs
2,
3). To measure 11-cis
and all-trans retinals, retinoids were extracted by the formaldehyde
method (Suzuki et al., 1988
;
Okajima and Pepperberg, 1997
)
as well as by the hydroxylamine method
(Groenendijk et al., 1980
)
before being analyzed on isocratic HPLC (same HPLC system except eluting at 1%
dioxane/hexane at 2 ml min-1). Both methods yielded similar
results. 11-cis retinyl esters and retinols were monitored at 318 nm,
and all-trans retinyl esters and retinols were monitored at 325 nm.
Retinals were monitored at 365 nm. Retinoids were identified by comparison of
their retention times to authentic standards, and their online photodiode
array UV spectra. Quantification was performed by comparing peak areas of
retinoids with those from calibration curves obtained from authentic standards
(Waters Millennium Software).
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Results |
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By contrast, Fig. 1B shows that the primary isomer in the light- and dark-adapted RPE is all-trans retinyl ester (peak II with a retention time of 6.5 min, monitored at 325 nm; Fig. 2B). A photodiode array spectrum shows that peak II has an absorption maximum of 325 nm, with an inflection at 323 nm, which is consistent with the spectrum of pure all-trans retinyl ester. Peak II also co-eluted with authentic all-trans retinyl palmitate, further confirming the identity of this HPLC component. Light exposure resulted in a 3-fold increase in the height of peak II, suggesting that light significantly increased the quantity of this all-trans retinyl ester in the chicken RPE.
When the same experiment was repeated by sampling at multiple time points under light and dark conditions (see Fig. 1C,D), the 11-cis retinyl ester pool in the retina increased nearly 6-fold in 2 h (open circles, Fig. 1C), while subsequent dark adaptation induced a rapid depletion of this pool to its original level within 1 h. Meanwhile, the all-trans retinyl ester pool in the retina remained relatively low and unchanged (filled circles, Fig. 1C). In distinctive contrast, upon illumination, the all-trans retinyl ester pool in the RPE increased about 3-fold (filled circles, Fig. 1D) while the 11-cis retinyl ester pool remained low (open circles, Fig. 1D). During the dark phase of these experiments, the rate of depletion of all-trans retinyl esters in the RPE was significantly lower than the depletion rate of the 11-cis retinyl esters in the retina (compare Fig. 1C,D).
A summary of measured rates of accumulation and depletion of the retinyl ester pools in the retina and RPE is shown in Table 1. During light and dark adaptation, the 11-cis retinyl ester pool in the retina accumulated (1.17 nmol h-1) and was depleted (-2.45 nmol h-1) more rapidly than the all-trans retinyl ester pool in the RPE, which increased during light adaptation at 0.32 nmol h-1 and decreased during dark adaptation at -0.27 nmol h-1. This suggests that the 11-cis retinyl ester pool was preferentially utilized for chromophore regeneration in dark conditions.
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When monitored at wavelengths of 325 nm and 318 nm, small amounts of additional 11-cis and all-trans retinyl esters (i.e. other than 11-cis and all-trans retinyl palmitates) were noted. Although they constituted a small portion of the total retinyl esters (i.e. less than 10% total), it would be appropriate to include them in our subsequent experiments to study the change in retinyl esters with time. Therefore, retinoid extracts were first saponified and the released retinols were measured isocratically by HPLC (as explained in the Materials and methods). Results from gradient HPLC in previous experiments did not reveal any significant level of free retinols in the hexane extract prepared from light- or dark-adapted retina and RPE.
Change in retinoids in isolated retina
To demonstrate that the 11-cis retinyl ester pool in the retina
was not derived from retinoids in the RPE, we first dissected dark-adapted
retina free of RPE. Retinae suspended in avian buffer were then exposed to
strong bleaching conditions (2500 lux from two 90 W halogen bulbs) for 0.5 and
1.0 h. Within 30 min of bleaching, the 11-cis retinyl ester pool
increased 3-fold from
0.07 to 0.26 nmol mg-1 while the
11-cis retinal pool decreased from
0.18 to 0.04 nmol
mg-1, indicating that
80% of all visual pigments were bleached
in the isolated retinae in vitro
(Fig. 2). This provides strong
evidence to suggest that bleaching of visual pigment (i.e. decrease in
11-cis retinal) resulted in an increase in the accumulation of
11-cis retinyl esters in the retina. The accumulation of
11-cis retinyl ester upon bleaching, rather than all-trans
retinyl ester, also suggests strongly that an isomerase activity is present in
the isolated retina. All-trans retinol was stable during dark and
light conditions at
0.07 nmol mg-1 while all-trans
retinyl ester increased from
0.01 to 0.1 nmol mg-1 after 1 h
(Fig. 2). The accumulation of
these all-trans retinoids may be the result of an impaired retinol
dehydrogenase (to convert all-trans retinal to all-trans
retinol) in the isolated retinae and the lack of RPE to which
all-trans retinol can be transferred and esterified. Nevertheless,
data from these experiments on isolated retinae clearly show that
11-cis retinyl ester was generated from within the retina. However,
the type of retinal cells where 11-cis retinyl ester is synthesized
or stored remains to be determined.
Retinoid visual cycle in young chickens
To define the visual cycle in the cone-dominated chicken, we quantitatively
measured changes in the level of the 11-cis, all-trans
retinyl esters and retinals in the chicken retina and RPE at different
durations of light and dark adaptation
(Fig. 3). Retina was dissected
free of RPE after light and dark treatments (see Materials and methods). In
the fully dark-adapted state, we found an average of 0.56 nmol of
11-cis retinal in the retina. Because 11-cis retinal exists
in a 1:1 stoichiometric ratio with cone opsin, our data are consistent with
previous reports that the chicken retina contains
0.4-0.5 nmol of
photopigments (rhodopsin and iodopsin) per eye
(Wald et al., 1955
;
Bridges et al., 1987
). Within 5
min of light exposure, the amount of 11-cis retinal dropped from
0.5 nmol eye-1 to 0.23 nmol eye-1. After an
additional 15 min of bleaching, only 0.15 nmol of 11-cis retinal per
eye was recovered (i.e.
30% of the dark-adapted value). After 5 min of
dark adaptation, the original level of chromophore was restored (
0.5 nmol
eye-1). This regeneration is consistent with the kinetics of
bleached cone pigment in the human eye fovea, which regenerate 100% of cone
pigment within 3 min of dark adaptation after strong bleaching conditions
(fig. 5 in Mahroo and Lamb,
2004
). During light conditions, all-trans retinal
increased to
0.15 nmol eye-1 from dark adapted levels of
0.04 nmol eye-1 (Fig.
3B). This reflects a continuing active processing of bleached
chromophore to other retinoids throughout light conditions.
After 5 min of strong light conditions, the amount of 11-cis
retinyl ester in the retina increased from 0.67 to
1.5 nmol
eye-1. This pool steadily increased to
2.1 nmol
eye-1 after 20 min of light conditions. The chicken eye, therefore,
produced 11-cis retinyl esters in the retina at about 4 molar
equivalent to the amount of photopigment in the eye within this short time
period (see Discussion on explanation of molar excess). This pool declined
rapidly within the first 5 min of dark adaptation from
2.1 to
1.0
nmol eye-1. This depletion readily accounts for the net amount of
regenerated pigment in the same time window (
0.4 nmol 11-cis
retinal per eye).
A steady increase of the all-trans retinyl ester pool in the RPE
was also seen after 20 min of light adaptation (0.17 to
0.52 nmol
eye-1) (Fig. 3B).
Interestingly, the net depletion of this pool within the first 5 min of dark
adaptation (
0.27 nmol eye-1) cannot account for the increase
of 11-cis retinal (
0.4 nmol eye-1) during the same
time period, suggesting that photopigment regeneration must be dependent on a
retinoid supply from the 11-cis retinyl ester pool. It should also be
noted that a significant part of this all-trans ester pool may be
contributing to the rod visual cycle in chicken much to the same extent that
it contributes to the rod visual cycle in rat
(Dowling, 1960
).
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Discussion |
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An estimated bleaching rate of 0.1 nmol min-1 can be derived
from data obtained during the first 5 min of bleaching in our experiments with
light- and dark-adapted chicken (Fig.
3A). Assuming that this rate continues throughout light exposure,
the amount of released chromophore is 2.0 nmol in 20 min. The
11-cis and all-trans retinyl esters in the retina increase a
net 2.0 nmol during this same time period. This suggests that the
11-cis retinal released from pigment bleaching can account for the
accumulation of retinyl esters.
Data from our work provides an estimated
(i.e. 50% of
the total time to reach reaction maximum) of 2.5 min for 11-cis
retinal regeneration in the chicken retina
(Fig. 3A). A
of
1 min
for the regeneration of human fovea photopigment (after a strong bleach) can
be deduced from the work of Mahroo and Lamb
(2004
). Given that the
is
6 min
for rod pigment regeneration (fig. 9 in
Lamb and Pugh, 2004
), our data
are consistent with the kinetics of cone regeneration. It is probable that our
estimated
of
2.5 min for chicken retinal chromophore regeneration deviates from
1 min
(for human cone pigment regeneration) because our data are derived from a
bleach of the mixed chicken photoreceptor population (i.e. 30-40% rod and
60-70% cone; see Meyer and May,
1973
), as opposed to the 100% fovea cone photoreceptor population
used in Mahroo and Lamb's studies (Mahroo
and Lamb, 2004
).
The newly identified visual cycle enzymatic activities (retinol isomerase,
11-cis retinyl ester synthase and 11-cis retinol
dehydrogenase) in the cone-dominated retina
(Mata et al., 2002), their
specific locale(s) and their relationship to the kinetics described here
should be the subject of future investigations. Das and colleagues were the
first to suggest that Müller glia may contribute substantially to
chromophore regeneration by demonstrating the isomerization of retinol by
chicken Müller cells in culture (1992). Cellular retinol binding protein,
cellular retinal binding protein
(Bunt-Milam and Saari, 1983
;
Eisenfeld et al., 1985
) and
retinal G-protein coupled-receptor (Pandey
et al., 1994
) are involved in vitamin A metabolism and have been
shown to be expressed in Müller cells. Müller apical processes
associate with the cell bodies of photoreceptors (for a review, see
Newman and Reichenbach, 1996
).
Interestingly, cones, but not rods, can transport 11-cis retinal from
the cell body to the outer segment (Jin et
al., 1994
). Is it possible that Müller glia isomerize retinol
and provide an 11-cis retinoid to the cone inner segment for
transport to the cone outer segment (COS)? If this is the case, Müller
glia may acquire additional chromophore precursor from vascular tissue through
end feet processes. This mechanism would be somewhat analogous to the RPE
obtaining vitamin A from the choroidal capillaries and could possibly account
for the excess retinoids in the retina (4 molar equivalent of retinyl esters
to photopigment) observed during light adaptation of the chicken retina.
Recently, the isomerization of all-trans retinyl ester in the RPE
has been implicated to be tightly controlled by RPE65, a protein essential to
this process (Redmond et al.,
1998). Soluble (sRPE65) and palmitoylated (mRPE65) isoforms of
RPE65 have been identified in the RPE (Ma
et al., 2001
). RPE65 has been shown to bind both
all-trans retinyl esters (Ma et
al., 2001
) and all-trans retinol
(Xue et al., 2004
).
Hypothetically, Xue et al.
(2004
) have suggested that, in
light conditions, the influx of all-trans retinol in the RPE is
shuttled to LRAT by sRPE65, where it is immediately esterified to produce
all-trans retinyl palmitate. All-trans retinyl palmitate may
donate acyl groups via LRAT to sRPE65, thereby generating mRPE65,
which can facilitate the isomerization reaction and generate 11-cis
retinol. In the retina, RPE65 protein is expressed in the mammalian and
amphibian COS but is absent in rods (Ma et
al., 1998
; Znoiko and Crouch,
2002
). This suggests that RPE65 may be serving one of its proposed
functions in the COS. The LRAT transcript and protein are absent from chicken
retina (Mata et al., 2005
).
Since mRPE65 contains a higher propensity to donate acyl moieties for
11-cis retinol over all-trans retinol via LRAT
(Xue et al., 2004
), it may
also provide this function in cone photoreceptors via 11-cis
ester synthase. Hypothetically, 11-cis retinol supplied by
Müller glia could be stored as 11-cis retinyl ester in the COS
during constant light conditions. Conversely, if sRPE65 is in the COS, it may
be shuttling retinol to another location for isomerization. Further studies
are needed to address these questions.
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Acknowledgments |
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