(Investigative Ophthalmology and Visual Science. 2002;43:564-572.)
© 2002
by The Association for Research in Vision and Ophthalmology, Inc.
Retinal Melatonin Production: Role of Proteasomal Proteolysis in Circadian and Photic Control of Arylalkylamine N-Acetyltransferase
P. Michael Iuvone1,
Audra D. Brown1,
Rashidul Haque1,
Joan Weller2,
Jolanta B. Zawilska1,
Shyam S. Chaurasia1,
Minhui Ma1 and
David C. Klein2
1 From the Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia; and the
2 Section on Neuroendocrinology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, Bethesda, Maryland.
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Abstract
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PURPOSE. Dynamic day–night changes in melatonin synthesis are regulated by
changes in the activity of serotonin N-acetyltransferase
(arylalkylamine N-acetyltransferase [AA-NAT]).
Similarly, a light-induced decrease in AA-NAT activity at
night rapidly suppresses melatonin synthesis. The purpose of the
current study was to test the hypothesis that in vivo changes of AA-NAT
activity in chicken retina homogenates parallel changes in AA-NAT
protein. This led to examination of the role of proteasomal proteolysis
in the regulation of retinal AA-NAT activity and protein levels.
METHODS. Chickens, entrained to a 12-hour light–12-hour dark cycle, were
assessed under various lighting conditions, in some cases after in vivo
intravitreal administration of the protein synthesis inhibitor
cycloheximide or lactacystin, an inhibitor of the 20S proteasome.
Tissue homogenates were prepared, AA-NAT enzyme activity was measured,
and immunoreactive protein was estimated by Western blot using an
anti-chicken AA-NAT1-21 serum.
RESULTS. The abundance of AA-NAT protein in both the retina and pineal gland
exhibited a daily rhythm that was statistically indistinguishable from
that of AA-NAT’s activity measured in tissue homogenates. Acute
exposure to light at night rapidly decreased AA-NAT protein and
activity in a parallel fashion. Administration of cycloheximide at
night decreased retinal AA-NAT activity in darkness and enhanced the
effect of light. The light-evoked suppression of retinal AA-NAT protein
and activity was blocked by intravitreal injection of lactacystin,
which also was found to increase AA-NAT activity, either at night or
during the daytime.
CONCLUSIONS. AA-NAT activity measured in tissue homogenates reflects the steady
state level of enzyme protein. AA-NAT protein in the retina turns over
rapidly, reflecting a balance of de novo synthesis and proteasomal
proteolysis. The suppressive effects of light at night are due
primarily to enhanced AA-NAT proteolysis.
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Introduction
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Melatonin is a neurohormone produced by retinal
photoreceptors and pinealocytes.1
2
3
4
In most vertebrate
species, the synthesis of melatonin in these tissues markedly increases
at night, reflecting the action of lighting and circadian
clocks.5
6
7
Retinal melatonin acts locally to promote dark
adaptation and to regulate various aspects of circadian retinal
physiology.6
8
Pineal-derived melatonin is secreted into
the circulation to act at central and peripheral target tissues to
control circadian physiology.9
In addition to the
well-defined circadian control of melatonin biosynthesis, mechanisms
have evolved to ensure that melatonin acts strictly as a signal for
darkness. As a result, unexpected light exposure at night rapidly
reduces the synthesis and levels of melatonin.10
11
12
The rate of formation of melatonin is regulated in part by the activity
of serotonin N-acetyltransferase (arylalkylamine
N-acetyltransferase [AA-NAT]; Enzyme Commission
[EC] no. 2.3.1.87), the penultimate enzyme in the melatonin
biosynthetic pathway. AA-NAT activity in retina and pineal gland of
most species examined undergoes robust circadian rhythms that generally
parallel the changes of melatonin synthesis.7
Recent
reports indicate that a major factor regulating rhythmic and
light-induced changes in activity is the steady state level of AA-NAT
protein, which reflects both synthesis and
degradation.13
14
15
16
Growing evidence indicates that both
processes may be subject to regulation and that there are surprising
and unpredictable species-dependent differences in the relative
importance of each mechanism, making analysis of each of special
importance.7
Synthesis is a reflection of AA-NAT mRNA levels. These are relatively
constant in the pineal glands of ungulates and some fish, but exhibit a
nearly 10-fold increase in the avian retina and pineal3
and a nearly 100-fold nocturnal increase in rodents.17
18
In contrast to these species-dependent differences in the
regulatory role of AA-NAT mRNA, in vitro studies of pineal glands
indicate that proteasomal proteolysis may play an important role in
circadian and photic regulation of AA-NAT in all
species.13
14
15
16
The in vivo regulation of the steady state levels of AA-NAT
protein in the avian retina and pineal gland has not been examined.
These tissues are of special value as models of vertebrate circadian
systems, because they contain complete circadian systems, including a
circadian clock, a clock entrainment mechanism, and mechanisms for
coupling the clock and visual transduction to melatonin
biosynthesis.3
19
20
In contrast, the mammalian pineal
gland relies on a clock located in the suprachiasmatic nucleus and a
photodetector located in the retina for circadian and photic input,
respectively.7
The circadian rhythms in AA-NAT activity in the chicken retina
and pineal gland in vivo reflect in part a circadian rhythm in AA-NAT
mRNA levels.3
However, this does not appear to fully
explain changes in AA-NAT activity, because AA-NAT activity and mRNA do
not change in parallel under all circumstances. For example, in diurnal
lighting the amplitudes of the activity rhythms are higher than those
of mRNA abundance, but under conditions of constant lighting they are
remarkably similar.3
In addition, light exposure at night
suppresses AA-NAT activity but not AA-NAT mRNA levels.3
These observations indicate that AA-NAT activity in the pineal gland
and retina is regulated by mechanisms other than or in addition to
AA-NAT mRNA levels. A likely explanation is that activity in vivo is a
reflection of the abundance of AA-NAT protein and that this is
regulated by proteasomal proteolysis. This proposal is supported by the
results of in vitro studies of chicken pinealocytes.15
This was examined in the in vivo study described herein, in which
chicken retinal and pineal AA-NAT activity and protein were examined.
These studies provide the first evidence for light-evoked proteasomal
proteolysis of AA-NAT in retinal photoreceptor cells. A preliminary
report of some of these data has been presented in abstract
form.21
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Methods
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Animals
Male White Leghorn chickens (Gallus domesticus) were
acquired (HyLine International, Covington, GA) on the day of hatching
and maintained in heated brooders in a 12-hour light–12-hour dark
cycle, with lights on from zeitgeber time (ZT) 0 to ZT 12. They were
used in experiments at 2 to 3 weeks of age. All manipulations of
chickens and tissues (before freezing) under conditions of darkness
were performed under dim red light (No. 92 filter; Eastman Kodak,
Rochester, NY). Chickens were killed by decapitation. The retinas and
pineal glands were rapidly dissected and frozen on dry ice. For
intravitreal drug administration, chickens were anesthetized with
halothane. Drugs were delivered into the vitreous cavity by injection
through a 30-gauge syringe needle in a volume of 10 µL. Animal use
was in compliance with the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research.
Preparation and Characterization of Anti-chicken
AA-NAT(cAA-NAT)1-21
The immunogen used to raise
anti-cAA-NAT1-21 serum (antiserum 2992) was
prepared by conjugating synthetic cAA-NAT1-21-C
(Research Genetics, Huntsville, AL) to injectible maleimide-activated
keyhole limpet hemocyanin (Pierce Chemical Co., Rockford, IL). The
immunogen was injected into rabbits (Covance, Vienna, VA), and titer
was monitored by dot blot, using cAA-NAT1-21-C
immobilized on polyvinylidene difluoride (PVDF) membrane (Immobilon-P;
Millipore, Bedford, MA). Crude antiserum 2992 was immunopurified using
AA-NAT1-21-C immobilized on PVDF
membrane.22
Antibody was eluted with 0.1 M acetic acid (pH
2.85) containing 0.1% bovine serum albumin (BSA; Intergen Corp.,
Purchase, NY).
Sample Preparation and Assays
Retina and pineal gland were homogenized in 75 and 37.5 µL,
respectively, of 0.25 M potassium phosphate (pH 6.5) containing 1.33 mM
acetyl coenzyme A, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10
µg/mL leupeptin, and 1 mM NaF. Homogenates were centrifuged at
40,000g for 1 hour at 4°C. The supernatant fraction of
each sample was used for measurement of total soluble
protein,23
AA-NAT activity24
and AA-NAT
protein by Western blot. Unless noted otherwise, 100 µg retinal
protein and 10 µg pineal protein were separated by SDS-PAGE
electrophoresis on 14% gels and transferred overnight at 160 mA to
PVDF membranes. Blots were incubated with a 1:200 dilution of
affinity-purified antiserum 2992 overnight at 4°C, followed by a 1-
to 2-hour incubation with secondary horseradish peroxidase–coupled
goat anti-rabbit IgG (Kirkegaard & Perry, Gaithersburg, MD).
Immunoreactive proteins were detected by enhanced chemiluminescence
(Amersham, Arlington Heights, IL) and analyzed by video densitometry by
computer (Lynx software; Applied Imaging, Newcastle-upon-Tyne, UK). For
semiquantitative analysis, blots were stripped and reprobed with a
monoclonal antibody to actin (Sigma Chemical Co., St. Louis, MO); the
density of the AA-NAT band was divided by that of the actin band and
normalized to the ratio at either ZT 18 or ZT 20, as specified in the
figure legends. In the experiment examining the effects of lactacystin,
AA-NAT blots were stripped and reprobed with a polyclonal antibody to
ubiquitin (Sigma Chemical Co.), before detection of actin.
Data Analysis
Data are expressed as the mean ± SEM. For statistical
comparisons of AA-NAT activity with AA-NAT–actin protein ratios,
activity measurements were normalized to the mean activity at ZT 18 or
ZT 20, as for the AA-NAT–actin protein ratios. The normalized data
were then analyzed by a two-factor ANOVA: factor 1, time of day or
treatment; factor 2, measurement (activity versus protein). All other
multiple comparisons were made with a one-way ANOVA with
Student-Newman-Keuls test.
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Results
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Characterization of Anti-cAA-NAT1-21
The specificity of anti-cAA-NAT1-21 (2992)
was examined by Western blot analysis, using crude (1:20,000) or
immunopurified (1:200) antiserum. Both preparations detected a single
strong 23-kDa band of immunoreactive protein in extracts of chicken
pineal glands obtained during the middle of the night (ZT 18; Figs. 1A 1B
). The density of the immunoreactive band was markedly lower in blots
of extracts obtained in the daytime (ZT 6). A positive signal was not
detected by the crude antiserum if it was first exposed to
cAA-NAT1-21-C immobilized on PVDF membrane (Fig. 1A)
. A 23-kDa immunoreactive protein band was also observed in extracts
of retina, but not in other central nervous system tissues (Fig. 1B)
:
heart, liver, kidney, intestine, or skeletal muscle (data not shown).
These findings establish the specificity of
anti-cAA-NAT1-21 (2992) for use in immunoblot
applications.
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Figure 1. Characterization of anti-cAA-NAT1-21 immunoreactivity.
Soluble proteins of tissue homogenates were subjected to SDS-PAGE,
transferred to PVDF membranes, and probed with
anti-AA-NAT1-21 antiserum or affinity-purified antibody.
(A) The crude antiserum and the affinity-purified antibody
recognized a major protein band of  23 kDa in nighttime extracts of
pineal gland. The apparent molecular weight of the protein corresponds
to the molecular weight predicted from the deduced amino acid sequence
of chicken AA-NAT. The intensity of the immunoreactive band was much
greater in nighttime extracts than in daytime extracts. The band was
absent from control blots probed with antiserum that was preincubated
with AA-NAT1-21 peptide. (B) The
immunoreactive protein was observed in nighttime extracts of retina and
pineal gland, but not in optic nerve, optic lobe, forebrain,
hypothalamus, or cerebellum. The immunoreactive protein was not
detected in blots of heart, liver, kidney, intestine, or skeletal
muscle protein (data not shown).
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Daily Rhythms of AA-NAT Protein and Activity in Retina and Pineal
Gland
AA-NAT activity was low during the daytime and high at night in
retinas and pineal glands of chickens maintained in a 12-hour
light–12-hour dark lighting regimen (Fig. 2)
. Both AA-NAT protein and activity change in parallel after a 24-hour
pattern, with high levels at night. Two-factor ANOVA indicated a
significant effect of time (retina, P < 0.01; pineal
gland, P < 0.01) but not of measurement (protein
versus activity) and no significant interaction of time and
measurement. Thus, the rhythms of AA-NAT protein in both tissues are
statistically indistinguishable from those of AA-NAT enzyme activity.
In this experiment, as in all subsequent experiments, the density of
the AA-NAT protein band in the immunoblots was normalized to that of
ß-actin. Representative ß-actin immunoblots are shown in
Figure 2 . ß-Actin immunoreactivity did not exhibit
significant changes on a daily basis or as a function of other
experimental manipulations (data not shown).
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Figure 2. Daily rhythms of AA-NAT protein and activity in retina (A)
and pineal gland (B). Subjects were housed in a 12-hour
light–12-hour dark cycle, with lights on from zeitgeber time (ZT) 24/0
to 12. Here and in Figure 3
, the open bar at the
x-axis represents time in light, and the filled
bar reflects time in darkness. Groups of retinas and pineal glands
were sampled at the times indicated for AA-NAT immunoreactive protein
and enzyme activity. Retinas and pineal glands corresponding to ZT 24
were dissected just before lights on, whereas those dissected at ZT 12
were collected in light, immediately before the time of lights off.
Top: representative Western blot of
anti-cAA-NAT1-21 immunoreactivity and ß-actin
immunoreactivity. Bottom: AA-NAT enzyme activity and
semiquantitative analysis of AA-NAT protein. AA-NAT protein is
expressed as the ratio of the density of the AA-NAT–immunoreactive
band to that of the actin-immunoreactive band, normalized to the ratio
at ZT 20. Immunoreactive protein and enzyme activity in both tissues
were rhythmic, and the rhythms of AA-NAT protein and activity for each
tissue were statistically indistinguishable. n = 4/group
(except retina ZT8, where n = 3). Two-factor ANOVA indicated
a significant effect of time (retina, P < 0.001;
pineal gland, P = 0.001) but not of measurement
(retina, P = 0.701; pineal gland, P =
0.237) and no significant interaction of time and measurement (retina,
P = 0.142; pineal gland, P = 0.681).
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To determine whether the rhythm of AA-NAT protein levels is driven by a
circadian clock, retinal AA-NAT enzyme activity and protein were
measured at midday and midnight for 2 days in constant darkness (Fig. 3)
. AA-NAT protein level and enzyme activity showed significant circadian
rhythmicity (P < 0.01) and fluctuated in parallel.
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Figure 3. Circadian rhythm of retinal AA-NAT protein and activity. Subjects
housed in a 12-hour light–12-hour dark cycle were subsequently exposed
to constant darkness for 54 hours. Groups of retinas were sampled at
the times indicated for AA-NAT immunoreactive protein and enzyme
activity. Top: representative Western blot analysis of
anti-cAA-NAT1-21 immunoreactivity. Bottom:
AA-NAT enzyme activity and semiquantitative analysis of AA-NAT protein.
AA-NAT protein is expressed as the ratio of the density of the
AA-NAT–immunoreactive band to that of the actin-immunoreactive band,
normalized to the ratio at ZT 19 of the first day in darkness.
Immunoreactive protein and enzyme activity levels followed a 24-hour
rhythmic pattern, and these rhythms for each tissue were statistically
indistinguishable. n = 5/group. Two-factor ANOVA
indicated a significant effect of time (P < 0.001)
but not of measurement (P = 0.694) and no
significant interaction of time and measurement (P = 0.961).
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Effects of Light Exposure at Night on AA-NAT Protein and Activity
Exposure to bright white light in the middle of the night (ZT 18)
suppressed AA-NAT protein levels and enzyme activity in a parallel
fashion in both retina (P < 0.01) and pineal gland
(P < 0.01; Fig. 4
). Protein levels and enzyme activity were maximally suppressed after 30
minutes of light exposure. This rapid disappearance of AA-NAT protein
could be due to an abrupt inhibition of AA-NAT synthesis if the protein
has a constitutive short half-life, or it could be due to accelerated
enzyme degradation in response to light.
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Figure 4. Acute light exposure at night decreased AA-NAT protein and activity.
Retinas (A) and pineal glands (B) corresponding
to ZT 18 were dissected at midnight in darkness (under dim red light).
Other groups of animals were exposed to bright white light (30
µW/cm2 reflected from a white surface at cage
level) beginning at ZT 18 for 0.5 or 1 hour, whereas another group
remained in darkness. Top: representative Western blot of
anti-cAA-NAT1-21 immunoreactivity.
Bottom: AA-NAT enzyme activity and semiquantitative analysis
of AA-NAT protein. AA-NAT protein is expressed as the ratio of the
density of the AA-NAT-immunoreactive band to that of the
actin-immunoreactive band, normalized to the ratio at ZT 18. Light
exposure significantly reduced both enzyme activity and protein.
n = 4/group. Two-factor ANOVA: retina, time,
P < 0.001; measurement, P = 0.482;
time x measurement, P = 0.849); pineal gland,
time, P < 0.001; measurement, P =
0.098; time x measurement, P = 0.444.
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Effect of Cycloheximide
In an attempt to distinguish between these possibilities,
the effects of inhibition of protein synthesis were compared with those
of light exposure on retinal AA-NAT protein and activity (Fig. 5)
. Cycloheximide (100 µg) was injected into the vitreous at ZT 17.5
under dim red light. Control eyes were injected with vehicle
(H2O). This dose of cycloheximide completely
suppressed the nocturnal increase of AA-NAT activity and tryptophan
hydroxylase activity in the chicken retina.25
Thirty
minutes after cycloheximide injection, AA-NAT protein and activity were
reduced to 69% and 61% of the ZT 17.5 control. At this time, half of
the animals were exposed to light, whereas the others remained in
darkness until death at ZT 18.5. During these subsequent 30 minutes in
darkness, protein level and enzyme activity continued to decrease. At
ZT 18.5, the levels were 43% and 47% of control, respectively. This
observation is consistent with AA-NAT’s having a short half-life in
darkness. Light exposure of control animals during the 30-minute period
from ZT 18 to 18.5 decreased enzyme protein and activity to 24%
and 47% of the ZT 17.5 control. Of special note was the
observation that the combined treatments of cycloheximide and light had
an additive effect, reducing enzyme protein and activity to 8% and
14% of control. Thus, in the absence of protein synthesis, light
reduced AA-NAT protein levels, indicating that illumination enhances
the degradation of AA-NAT.
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Figure 5. Additive effects of light and cycloheximide (CHX) on AA-NAT activity.
Retinas of uninjected control eyes were dissected in darkness (D) at ZT
17.5. Anesthetized chicks were injected intravitreally with 100 µg
CHX at ZT 17.5. Separate groups of chicks were injected with
H2O (Veh). Retinas were dissected at ZT 18 in D. Half of
the chicks remained in D, whereas the others were exposed to light (L)
for 0.5 hours. Retinas were dissected at ZT 18.5 and assayed for AA-NAT
protein and activity. (A) Representative AA-NAT immunoblot.
(B) Semiquantitative analysis of AA-NAT protein. AA-NAT
protein is expressed as the ratio of the density of
AA-NAT–immunoreactive band to that of the actin-immunoreactive band,
normalized to the ratio at ZT 17.5. (C) AA-NAT enzyme
activity. Light alone (P < 0.001) and cycloheximide
alone (P < 0.001) decreased AA-NAT activity at ZT18.5.
The effects of the two treatments were additive (light + CHX versus
dark + CHX at ZT 18.5, P < 0.001; light + CHX versus
light control at ZT 18.5, P = 0.0012). n
= 4 to 5/group. Two-factor ANOVA: treatment, P <
0.001; measurement, P = 0.290; treatment x
measurement, P = 1.084.
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Effects of Lactacystin on the Light-Evoked Suppression of AA-NAT
Protein and Activity
Proteasomal proteolysis has been implicated in the regulated
degradation of rat pineal AA-NAT.13
To investigate the
possible involvement of proteasomal proteolysis in the light
suppression of retinal AA-NAT, chickens were injected intravitreally
with the water-soluble inhibitor of the 20S proteasome lactacystin
(Fig. 6)
. Other aqueous-insoluble inhibitors could not be tested, because they
rapidly precipitated after injection into the vitreous. Lactacystin
inhibited light-evoked suppression of retinal AA-NAT activity in a
dose-dependent manner (P < 0.001), with a maximal
effect at 25 nmol/eye (Fig. 6A)
. At this dose, lactacystin completely
inhibited the effect of light on AA-NAT enzyme activity, which remained
stable in light-exposed retinas for at least 40 minutes (Fig. 6B)
.
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Figure 6. Proteasomal regulation of retinal AA-NAT activity. Lactacystin
inhibited the acute inhibitory effect of light. Dark-adapted chicks
were injected intravitreally at ZT 17 with lactacystin, a proteasome
inhibitor, and with vehicle (contralateral eye) and were exposed to
light for various times beginning at ZT 18. (A) Lactacystin
blocked the light-evoked decrease of AA-NAT activity in a
dose-dependent manner. Chicks were exposed to light for 20 minutes.
Doses of 25 and 50 nmol/eye significantly attenuated the effect of
light (light vehicle versus light lactacystin 25 nmol,
P < 0.001; light vehicle versus light lactacystin 50
nmol, P = 0.002). n = 4 to 5/group.
(B) Lactacystin, 25 nmol/eye, completely prevented the
effects of 20 and 40 minutes of light exposure (P <
0.001). n = 4 to 6/group.
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Lactacystin also significantly inhibited the light-evoked decrease of
AA-NAT protein immunoreactivity (Fig. 7)
. The effect of light on AA-NAT immunoreactivity surprisingly was only
partially inhibited by lactacystin, even though the drug completely
blocked the suppression of enzyme activity in the same retinas.
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Figure 7. Lactacystin inhibited the light-evoked decrease of AA-NAT protein in
retina. Lactacystin (25 nmol/eye) and vehicle (H2O) were
injected intravitreally at ZT 17. Chickens were subsequently exposed to
light for 20 minutes, beginning at ZT 18. Top:
representative Western blot for AA-NAT immunoreactive protein.
Bottom: AA-NAT enzyme activity and protein levels,
normalized to the dark control at ZT 18. Lactacystin completely
prevented the light-evoked decline of AA-NAT activity and partially
inhibited the light-evoked decrease of the AA-NAT–immunoreactive
protein band. n = 4/group. Two-factor ANOVA: treatment,
P < 0.001; measurement, P =
0.012; treatment x measurement, P = 0.051.
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To confirm that lactacystin inhibited proteasomal proteolysis under
these experimental conditions, the blots were reprobed with an
anti-ubiquitin antiserum (Fig. 8)
, because lactacystin is thought to act by blocking proteasomal
processing of ubiquitinated proteins.26
This
revealed the presence of both low-molecular-weight immunoreactive
ubiquitin, corresponding to free ubiquitin, and a high-molecular-weight
smear of polyubiquitinated proteins. Light exposure had no significant
effect on polyubiquitinated proteins (normalized ubiquitin/actin: dark
1.0 ± 0.4; dark
light 1.9 ± 0.9; n = 4).
Lactacystin, however, elicited a large increase of ubiquitin
immunoreactivity (4.7 ± 0.4; n = 4,
P < 0.01), consistent with inhibited proteolysis of
ubiquitinated proteins.
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Figure 8. Lactacystin increases the accumulation of ubiquitin-immunoreactive
proteins in retina. Lactacystin (25 nmol/eye) and vehicle
(H2O) were injected intravitreally at ZT 17. Chickens were
subsequently exposed to light for 20 minutes, beginning at ZT 18.
Retinal extracts from chicks exposed to light for 20 minutes at ZT 18
were subjected to immunoblot analysis with a polyclonal anti-ubiquitin
antiserum. D, dark at ZT 18; DL, 20-minute light exposure beginning
at ZT 18. Lactacystin treatment increased high-molecular-weight
immunoreactivity.
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Effects of Lactacystin on the Daily Rhythm of AA-NAT
Protein Level and Enzyme Activity
To assess the role of proteolysis in the daily rhythm of AA-NAT,
lactacystin was administered intravitreally to chicks maintained in a
12-hour light–12-hour dark cycle, 1 hour before death at midnight or
midday (Fig. 9)
. Lactacystin treatment increased both AA-NAT protein level and enzyme
activity at midnight in darkness (P < 0.01). In
contrast to the effect of lactacystin after acute light exposure at
night, the increase of immunoreactive protein was equivalent in
magnitude to the increase of activity. Treatment with lactacystin in
the middle of the day in light increased AA-NAT activity
(P < 0.001). The magnitude of the effect was
relatively small, consistent with evidence that synthesis of AA-NAT is
lower during the day, because AA-NAT mRNA is lower.
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Figure 9. Effect of lactacystin on the daily rhythm of retinal AA-NAT protein and
activity. Lactacystin (25 nmol/eye) and vehicle (H2O) were
injected intravitreally at ZT 5 in light and at ZT 17 in darkness.
Retinas were dissected 1 hour thereafter and assayed for AA-NAT protein
and activity. Top: representative Western blot shows
anti-cAA-NAT1-21 immunoreactivity. Bottom:
AA-NAT enzyme activity and semiquantitative analysis of AA-NAT protein.
AA-NAT protein was expressed as the ratio of the density of the
AA-NAT–immunoreactive band to that of the actin-immunoreactive band,
normalized to the ratio of the vehicle control at ZT 18. n
= 10/group. At night, lactacystin significantly increased
immunoreactive protein and enzyme activity in parallel, with no
significant differences between activity and protein levels. Two-factor
ANOVA: treatment, P < 0.001; measurement,
P = 0.427; treatment x measurement,
P = 0.429. During daytime, lactacystin
significantly increased AA-NAT activity (P <
0.001), but not that of protein (P = 0.095).
Two-factor ANOVA: treatment, P < 0.001;
measurement, P < 0.001; treatment x
measurement, P < 0.001.
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It is of interest to note that the lactacystin-induced increase in
AA-NAT immunoreactive protein during the day (in light) appeared to
have a lower magnitude than that of enzyme activity, similar to the
effect of lactacystin in light-exposed retinas at night. The increase
of immunoreactive protein elicited by lactacystin was not statistically
significant during the daytime (P = 0.095).
Furthermore, there was a significant effect of treatment (activity
versus protein) in the lactacystin-treated daytime retinas
(P < 0.001), but not in the vehicle-treated daytime
samples (P = 0.11).
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Discussion
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In the results of the present study, the daily fluctuations
of AA-NAT activity in chicken retina and pineal gland in vivo
correlated with fluctuations of the 23-kDa immunoreactive protein
AA-NAT. Furthermore, they demonstrated that the rapid suppression of
AA-NAT enzyme activity resulting from light exposure at night was
caused by a reduction of immunoreactive enzyme protein. These results
confirm and extend the findings of Zatz et al.,15
who
demonstrated a daily rhythm of AA-NAT protein abundance in cultured
chicken pinealocytes.
Zatz et al.15
examined AA-NAT protein abundance
using an immunoprecipitation assay with an antibody directed against
recombinant rat glutathione-S-transferase (GST)-AA-NAT.
AA-NAT protein was identified by autoradiography after phosphorylation
by cAMP-dependent protein kinase and
[32P]-adenosine triphosphate (ATP). The assay
detected two phosphorylated bands close to the predicted molecular
weight of AA-NAT that fluctuated in parallel. In the present study, an
antiserum against the N-terminal amino acid sequence of chicken AA-NAT
was used that detects AA-NAT in retina and pineal gland by Western
blot. In contrast to the assay of Zatz et al.,15
anti-cAA-NAT1-21 detected only a single major
protein band of 23 kDa in pineal extracts. The reason for this
discrepancy is unclear. AA-NAT contains two consensus phosphorylation
sites for cyclic nucleotide-dependent protein
kinases.3
7
27
Thus, the two radioactive protein bands
observed in the earlier study may reflect mono- and diphosphorylated
forms of the enzyme. Alternatively, the bands may represent intact and
partially degraded enzyme protein, because the
anti-cAA-NAT1-21 serum used in the present study
would not detect a partially degraded protein if the N-terminal 21
residues had been deleted. It is also possible that other
posttranslational modifications of this sequence influence the
immunodetection of AA-NAT.
The rhythm of chicken AA-NAT activity appears to reflect rhythmic
changes in the steady state levels of enzyme molecules, as indicated by
the parallel changes of enzyme protein and activity observed in the
present study. Two processes appear to contribute to the observed
rhythm in AA-NAT protein. As discussed in the following paragraph, one
is the clock-driven rhythm in AA-NAT mRNA, which is translated into a
rhythm in synthesis. The second is enzyme degradation, which is
regulated by light.
Unexpected light exposure at night rapidly suppresses AA-NAT
activity and melatonin levels in chicken retina and pineal
gland.12
In the current study, this rapid suppression was
caused by a decrease of enzyme protein. The suppression of enzyme
activity under these conditions was not accompanied by a comparable
decline in AA-NAT mRNA abundance,3
suggesting a
posttranscriptional site of regulation. This is supported by the
evidence that AA-NAT has a constitutively high turnover rate in
darkness, as indicated by the finding that inhibition of protein
synthesis at night in darkness caused AA-NAT activity and protein to
decrease. It is not clear, however, whether physiological regulation by
light involves inhibition of protein synthesis. This is not
unreasonable to consider, because the decrease in AA-NAT activity and
protein due to light treatment was similar to that produced by
inhibition of protein synthesis. However, it was also found that
cycloheximide treatment markedly enhanced the effects of light,
indicating that light does not act exclusively by inhibiting protein
synthesis. but rather it acts, at least to a large degree, by
accelerating AA-NAT degradation. The possibility that light also
inhibits translation remains open to further investigation.
The results of several experiments and our understanding of the
regulation of AA-NAT in other vertebrate systems are consistent with
the hypothesis that photic regulation of chick AA-NAT protein is due to
cAMP control of AA-NAT proteolysis. This is supported by several
observations. First, photoreceptor cAMP is regulated by light exposure,
which decreases levels of this second messenger.28
29
30
31
Second, cAMP inhibits the decline of AA-NAT activity in
cycloheximide-treated photoreceptor cells.32
Third, the
cAMP protagonist forskolin increases AA-NAT protein and activity in
chick pinealocytes,15
apparently due to reduced
destruction of AA-NAT. Fourth, as discussed earlier, when protein
synthesis is blocked, AA-NAT protein disappears, and forskolin can
prevent this,32
presumably by elevating cAMP. These
observations lead us to conclude that light and darkness act through
cAMP to regulate AA-NAT protein. Other observations indicate that cAMP
is probably acting through phosphorylation of the highly conserved PKA
sites in AA-NAT, at least one of which is known to be phosphorylated at
night in the ovine pineal gland.33
Proteasomal proteolysis has been implicated in the regulation of AA-NAT
protein turnover in pineal gland. Proteasome inhibitors increase AA-NAT
protein levels of cultured rat pineal cells and prevent the reduction
in protein levels caused by blocking ß-adrenergic
receptors.13
Similarly, treatment with proteasome
inhibitors increased AA-NAT protein levels and enzyme activity in
cultured chick pinealocytes.15
In the present study,
lactacystin, an inhibitor of the 20S proteasome, significantly
inhibited the light-evoked suppression of retinal AA-NAT protein
abundance and enzyme activity in vivo. Thus, light may stimulate
proteasomal degradation of AA-NAT in photoreceptor cells. Inhibition of
proteasomal degradation by treatment with lactacystin prevented the
light-evoked suppression of AA-NAT activity but did not increase it
significantly above the levels in dark-adapted retinas. The surprising
observation that light treatment partially decreased AA-NAT protein in
the presence of lactacystin indicates that under certain experimental
conditions AA-NAT protein and activity do not change in parallel.
Lactacystin also increased AA-NAT protein and activity at
midnight in darkness, consistent with the hypothesis that AA-NAT is
being synthesized and destroyed continually in darkness. The effect of
lactacystin on AA-NAT activity and protein in darkness was similar;
both increased in parallel. In contrast to this relationship,
lactacystin treatment in the light caused AA-NAT activity to increase
more than that of immunoreactive protein. This provides further
evidence that AA-NAT activity and protein do not change in parallel
under all conditions.
The reason for the loss of parallelism is not clear but could
involve a posttranslational modification of the enzyme protein that
changes immunoreactivity without changing catalytic activity. For
example, light exposure may lead to structural modification that does
not change activity but increases targeting to the proteasome through
modifications that reduce immunoreactivity. This would result in an
apparently greater preservation of activity than of immunoreactive
protein. Many proteins targeted for proteasomal degradation are
ubiquitinated at lysine residues near the N-terminal,26
and ubiquitin conjugation and degradation in rat retina are greater
under light-adapted than dark-adapted conditions.34
Ubiquitination at Lys-10 of AA-NAT may block immunoreaction with our
antibody, which was raised against N-terminal amino acids 1-21. It is
also possible that the immunoreactive band of AA-NAT is a reflection of
both denatured and active protein and that both are always present in
the cell. Accordingly, lactacystin may have a more dramatic effect on
enzyme activity, because the assay measures only active protein,
whereas the Western blot assay detects both active and denatured
protein.
Recently, AA-NAT was shown to bind to the ubiquitous signaling protein
14-3-3.35
The binding of AA-NAT by 14-3-3 apparently
modulates its enzyme activity by stabilizing a region of the enzyme
involved in substrate binding.35
Although the role of this
interaction in photoreceptors remains to be investigated, it seems
reasonable to suspect that it may play a role in the activity and/or
stability of AA-NAT in the retina, and as a result, in melatonin
production in this tissue.
In conclusion, proteasomal proteolysis plays a prominent role in the
physiological regulation of retinal AA-NAT activity. The rapid
suppressive effect of light on AA-NAT activity and melatonin
biosynthesis is primarily a reflection of enhanced enzyme proteolysis.
|
Acknowledgements
|
|---|
The authors thank Pierre Greve for pilot studies on the AA-NAT
Western blot analysis.
|
Footnotes
|
|---|
Supported in part by NIH Grant ROI EY04864 (PMI).
Submitted for publication May 17, 2001; revised September 10, 2001;
accepted October 1, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: P. Michael Iuvone, Emory University,
Department of Pharmacology, 1510 Clifton Road NE, Atlanta, GA
30322-3090; miuvone{at}pharm.emory.edu
|
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Top
Abstract
Introduction
