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Section on Neuroendocrinology (J.F., K.M.G., J.L.W., S.L.C., D.C.K.), Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4480; Laboratoire de Neurobiologie Cellulaire (J.F.), Département des Neurosciences, CNRS-UMR 6558, F-86022 Poitiers Cédex, France; and Fish Physiology Department (B.R., G.C.), National Center for Mariculture, Oceanographic & Limnological Research Institute, Eilat 88112, Israel
Address all correspondence and requests for reprints to: David C. Klein, NIH 49–6A82, Bethesda, Maryland 20892-4480. E-mail: klein{at}helix.nih.gov
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In all vertebrates, melatonin production follows a circadian pattern that is controlled by melatonin rhythm generating systems that are typically composed of three basic elements, a photodetector, an endogenous clock and melatonin synthesizing machinery. In mammals, the melatonin rhythm generating system includes the retina (photodetector), suprachiasmatic nucleus (clock), and pineal gland (melatonin synthesis) (2). In contrast, in birds and fish, complete melatonin rhythm generating systems are located within individual photoreceptor cells in the pineal gland and retina (4). In most cases, this includes a photodetector, circadian clock, and melatonin synthesis machinery; however exceptions exist. For example, trout melatonin rhythm generating systems appear to lack a functional circadian clock. In species with cellular melatonin rhythm generating systems, melatonin production persists on a circadian basis when the pineal glands are placed in culture (5, 6).
Large changes in melatonin production are typically associated with similar changes in the activity of the penultimate enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87), which controls the rate at which serotonin is converted to N-acetylserotonin (7, 8). In most vertebrates, only a single AANAT gene exists. However, in fish there are two AANAT genes, termed AANAT1 and AANAT2 (9, 10, 11). AANAT2 is the dominant form expressed in the pineal organ. AANAT1 is expressed predominantly in the retina, and is more similar to the AANAT in other vertebrate classes. AANAT1 acetylates indole- and phenyl-ethylamines equally well; AANAT2 strongly prefers indole-ethylamines.
The mechanisms controlling AANAT protein vary among vertebrates. For example, in some—but not all—cases changes in AANAT messenger RNA (mRNA) occur; in these species, low levels of AANAT mRNA determine when AANAT activity can increase. In most cases, mechanisms appear to exist at a post transcriptional level to control levels of AANAT protein (8, 12, 13).
In the pike pineal organ, the endogenous clock regulates changes in AANAT2 activity and AANAT2 mRNA (6, 10). Rhythmic changes in AANAT2 activity drive the rhythm in melatonin production. The translation of endogenously driven changes in AANAT2 mRNA into changes in AANAT2 activity is influenced by an exogenous signal –light. Light acts through two distinct pathways: one adjusts and resets the endogenous circadian clock and, as a result, the rhythm in AANAT2 mRNA and activity (6); this effect is of a gradual nature and is likely to involve shifting the rhythmic expression of clock genes (14). Light also acts rapidly, independently of the clock to suppress AANAT2 activity. This suppressive influence is in part responsible for low day levels of AANAT2 activity and for the light-induced suppression of enzyme activity that occurs when pike are exposed to a pulse of light at night. The importance of this mechanism is most obvious in trout, which, as indicated above, lack a pineal circadian clock and express the AANAT2 gene constitutively. In this case, light appears to be the dominant regulatory mechanism controlling day/night changes in AANAT activity and melatonin production (6).
The precise molecular details mediating the immediate effects of light on AANAT2 activity are poorly known. Studies in the rat, sheep, and chicken pineal organ indicate that AANAT activity and protein change in parallel on a daily basis. Studies of the rat and chicken pineal in culture indicate that AANAT protein is influenced by a cAMP-dependent mechanism that blocks destruction of AANAT protein via proteasomal proteolysis (12, 13). The high degree of homology between the different vertebrates AANATs, especially in the putative N-terminal region flanking the catalytic core of the molecule, suggests a similar mechanism might also operate in fish to control AANAT2 protein. However, regulation of fish AANAT2 protein has not been examined and it is not clear that mechanisms exist to control AANAT2 protein. In addition, second messenger control of fish pineal AANAT2 is still a matter of debate (6, 15). And, limited observations suggest that proteasomal proteolysis plays a minor role in controlling fish AANAT2 activity (15).
The present study focused on the question of whether AANAT2 protein in the fish pineal organ is regulated in a manner consistent with a role in regulating AANAT activity, as is the case in birds and mammals. Three fish species, pike, seabream and trout, were studied. Pike is a well studied nondomesticated fresh water species; the pike pineal gland contains a complete melatonin rhythm generating system, as is the case in the chicken (16). The seabream pineal organ also contains an endogenous clock; this species is of interest because it is of marine origin and is domesticated (17). In addition, it is abundantly available for analysis. A better understanding of melatonin production in this species might improve commercial breeding. Trout were used because AANAT2 activity is regulated by light without the mediation of an endogenous clock (18).
The results of the study indicate that there is a daily rhythm in the pike pineal gland in AANAT2 protein and that AANAT2 protein and activity change in a parallel manner in vivo and in vitro. Results from studies of all three species make apparent that regulatory systems are present in the fish pineal organ through which AANAT2 protein can be increased by elevation of cAMP or by inhibition of proteasomal proteolysis.
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Materials and Methods |
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Trout (Oncorhynchus mykiss, teleost; 300 to 1,000 g) of both sexes were obtained from a commercial breeder ("Pisciculture du Moulin de Pied de Lance," Vienne, France). Animals were killed soon after their arrival at the laboratory (1100 and 1130 h); their pineal organs were removed and placed in culture medium; all other conditions were as above.
Seabream (Sparus aurata, teleost; 200–300 g) of both sexes were obtained from the National Center for Mariculture at Eilat (Israel). The photoperiod was 13.75 L (0545–1930)/10.25D. They were killed early in the afternoon, and the pineal organs were collected and prepared as above.
Organ culture
The culture medium was RPMI 1640 supplemented as indicate before
(19). The pineal organs were cultured in 24-well culture
plates containing 0.5 ml culture medium. Temperature was maintained
constant at 22 C (seabream), 20 C (pike) or 12 C (trout). It has been
shown that these temperatures are optimal for the culture of these
organs (6). After removal, the pineal organs were cultured
for 24 h under photoperiodic conditions similar to the natural
ones (L = 1000 lux intensity). Experimental treatments were
initiated thereafter as detailed in Fig. 1
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Treatments of trout pineal organs were always initiated at 1200 h because in this species, exposure to darkness at any time of the light/dark (LD) cycle produces similar increases in pineal AANAT2 activity and melatonin secretion (20, 21). Treatments of pike and seabream pineal organs were initiated at the onset of darkness, because the circadian oscillator that governs AANAT2 activity and melatonin secretion prevents a dark-induced rise in AANAT2 activity during the day and second half of the night (22, 23). Following the indicated treatments, organs were collected under fluorescent white light (light samples) or dim red light (dark samples) and frozen on dry ice. Treatments are described in the text and legends.
Assay of AANAT1 and AANAT2 activities
AANAT1 and AANAT2 activities were determined following
modification (9, 11) of a published radiochemical
procedure (24). To measure AANAT2 each pineal organ was
sonicated in 50 µl of assay buffer (0.2
M, pH 6 phosphate buffer), and the
homogenate was added to a 50 µl buffer solution containing tryptamine
(10 mM), acetyl-CoA (0.5 mM) and
[3H]-acetyl-CoA (4 mCi/mmol). Incubation was
for 60 min at 12 C (trout), or 20/22 C (pike, seabream).
N-[3H]-Acetyltryptamine was
extracted into chloroform and radioactivity was determined. For the
estimation of retinal AANAT1 activity phenylethylamine was used as the
substrate, and the homogenates were incubated for 20 min at 37 C
(9).
Production of anti-AANAT2 antisera (3345)
pAANAT24–29 [Accession No.
AF034082] was conjugated to cationized BSA for use as an immunogen.
Polyclonal anti-pAANAT24–29 serum was raised in
a rabbit [#3345] (Covance Laboratories, Inc., Vienna,
VA). Antiserum was immunopurified by exposure to sections of blots
containing the immunizing peptide (pAANAT24–29);
the adsorbed antibodies were eluted with 0.1 N acetic acid,
pH 2.85, containing BSA (25).
Production of recombinant proteins
Recombinant glutathione S-transferase (GST) pike (p)AANAT2
(Accession No. AF034082, 10) and pAANAT1 (Accession No.
AF034084) fusion proteins were obtained after transformation and
expression in Escherichia coli strain BL21(DE3) pLysS. The
methods used have been described (26), except that cells
were grown overnight at 20 C after addition of
isopropylthiogalactopyranoside. After centrifugation, cells (from 1
liter) were resuspended in 50 ml lysis buffer (2x PBS, 10
mM dithiothreitol, 1 mM
EDTA) and stored at -80 C. Upon thawing, cells were lysed by
sonication at 4 C followed by centrifugation at 48,000 x
g for 45 min. A 5 ml sample of glutathione Sepharose 4B,
previously equilibrated in 1x PBS, was added to the supernatant, and
the slurry was mixed at 4 C for 1 h. The resin was then poured
into a 100 ml column (Bio-Rad Laboratories, Inc.) and
washed with 10 column volumes of buffer 2 (1x PBS, 0.5
M NaCl, 10 mM DTT, 1
mM EDTA) followed by 10 column volumes of buffer
3 (20 mM Tris [pH 7.5], 0.5
M NaCl, 10 mM DTT, 1
mM EDTA, 10% [wt/vol] glycerol). The protein
was eluted at room temperature using 10 mM
glutathione in buffer 3 (pH adjusted to 
8). Fractions containing the
fusion protein (judged by absorption at 280 nm) were combined and
dialyzed against buffer 3 containing 1 mM DTT.
This and subsequent steps were carried out at 4 C in buffers sparged
with helium. The GST-fusion protein was cleaved by adding 5 U thrombin
per mg protein, and the reaction was monitored by SDS-PAGE. After
cleavage, free GST and thrombin were removed by passage over a second
column consisting of 2.5 ml glutathione Sepharose 4B and 0.5 ml
benzamidine Sepharose 4B equilibrated in dialysis buffer. The recovered
protein (>90% pure) was dialyzed against 50 mM
sodium citrate (pH 6.5), 0.1 M NaCl, 10
mM DTT, 1 mM EDTA, 10%
(wt/vol) glycerol. The final yield from 1 liter cells was approximately
4 mg protein.
Western blots
Pineal proteins were resolved on pre-formed 14% Tris/glycine (1
mm) gels using the standard Novex polyacrylamine gel
electrophoresis protocol (Novex, Invitrogen,
Carlsbad, CA). The gels were run at 125 V for 2 h. Rainbow
standards (Amersham Pharmacia Biotech, Piscataway, NJ)
were used to determine the molecular weight of the proteins.
Before electroblotting, gels were equilibrated (5 min) in 10 mM [3cyclohexylamino]-1-propanesulfonic acid buffer, pH 11, containing 20% methanol and 0.1% SDS. The proteins were transferred in this buffer to Immobilon-P membrane (0.45 µm; Millipore Corp., Bedford, MA) with a semidry blotting following the manufacturers protocol system (Investigator Graphite Electroblotter system; Genomic Solutions; Chelmsford, MA). The transfer currents were 400 µA/cm2 (20 min), then 800 µA/cm2 (20 min) and finally 1200 µA/cm2 (45 min).
After transfer, the membranes were air dried and then blocked for 2 h in phosphate buffer saline (PBS, pH 7.4) containing 10% nonfat dry milk, 0.2% Tween-20 and 0.05% thimerosal (TPBS). They were then incubated overnight (18 h at room temperature) in the primary antibody solution (1/250 dilution in PBS containing 1 mg BSA fraction V, and 0.05% thimerosal). The membranes were then washed successively in TPBS (2 x 5 min) and PBS (2x 5 min) before a 1 h exposure to horseradish peroxidase conjugated goat antirabbit IgG (0.0083 µg/ml in TPBS containing 0.1% normal goat serum). The membranes were then washed successively in TPBS (3 x 10 min) and PBS (3 x 5 min) and immunodetection was performed using Luminglo. Immunopositive reactions on the blots were finally exposed to BIOMAX MR or Kodak XAR 5 films. The immunopositive bands were quantified using the ImageQuant program (Molecular Dynamics, Inc., Sunnyvale, CA) after the films were scanned.
Chemicals
Acetyl-CoA was from Roche Molecular Biochemicals
(Ingelheim, Germany). [3H]-acetyl-CoA, was from
NEN Life Science Products (Boston, MA).
Clasto-lactacystine (ß-lactone), lactacystine, MG132, proteasome
inhibitor I, calpain inhibitor II were from Calbiochem (La
Jolla, CA). Synthetic pAANAT24–29 was made by
Research Genetics, Inc. (Huntsville, AL). RPMI 1640 was
from Life Technologies, Inc.
(Rockville, MD). Nonfat dry milk and glutathione
Sepharose 4B were from Bio-Rad Laboratories, Inc.
(Hercules, CA). Goat antirabbit IgG conjugated to horseradish
peroxidase and Luminglo were from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Normal goat serum was from
Pierce Chemical Co. (Rockford, IL). All other products
were from Sigma (St. Louis, MO).
Statistics
All experiments were run in duplicate or triplicate. Variability
did not exceed 10%. Values were compared using the Student’s
t test, or by ANOVA followed by Tukey’s comparison of means
(Prism software, GraphPad Software, Inc., San Diego,
CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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); the signal
generated by the recombinant pAANAT2 protein had the same mass (Fig. 2A). An approximately 26-kDa band was also detected in the retina,
albeit at a distinctly less intense level (Fig. 2B). Bands of this mass
were not detected in other tissues, although nonspecific immunoreactive
bands of higher mass were apparent, including two strong at
approximately 30–35 kDa in the pituitary (Fig. 2B). Based on this
pattern of expression and the predicted mass of pike AANAT2 protein (24
kDa), the approximately 26 kDa band of protein was used as an indicator
of the amount of pAANAT2 protein. The observation that there is a
difference between the predicted and apparent masses of both the
expressed and recombinant pAANAT2 protein suggests that this is due to
an inherent feature of the protein.
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). In addition, exposure to 1 h of
light at night decreased the strength of the signal (Fig. 3B); exposure
to darkness at midday failed to elevate AANAT2 activity and protein to
night levels (Fig. 3B), consistent with the known involvement of a
pineal clock in the regulation of pAANAT2 mRNA and activity as
discussed (27, 19). In all cases, changes in AANAT2
activity were paralleled by similar changes in AANAT2 protein, as has
been seen in other species (8, 12).
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).
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). In addition,
midnight illumination of dark-adapted organs induced a dramatic
decrease of both pAANAT2 activity and pAANAT2 protein levels. As shown
previously (6), agents that increase intracellular cAMP
levels, such as the adenylate cyclase activator forskolin, increase
pAANAT2 activity. Here we found that in vitro treatments
with forskolin during the day increased pAANAT2 protein, and that
activity and protein changed to a similar degree (Fig. 4). Similar
results were obtained by treatment with isobutylmethylxanthine, an
inhibitor of cAMP phosphodiesterase (not shown).
To test the hypothesis that AANAT2 protein is destroyed by proteasomal
proteolysis in the fish pineal gland, we first examined the effects of
an inhibitor of proteasomal degradation, MG132. Treatment with MG132
increased both pAANAT2 protein and activity. The effects of MG132
appeared to be larger than those due to forskolin treatment (Fig. 4).
This was most evident when drug treatments were done under
illumination. In dark-adapted pineal organs, treatment with both MG132
and forskolin totally prevented the suppressive effects of light on
pAANAT2 activity and protein. It is notable that forskolin
and/or MG132 had similar effects on AANAT2 protein and AANAT2 activity
in all three different lighting conditions, providing convincing
evidence that either agent enhances AANAT protein levels, and that this
does not appear to be an effect that is dependent upon environmental
lighting (Fig. 4).
Inhibitors of proteasomal proteolysis increase AANAT2 activity in
cultured pike, trout and seabream pineal organs
The above effects of MG132 on AANAT2 protein provide direct
evidence that the fish pinealocyte has the intracellular machinery
required to destroy AANAT2 protein by proteasomal proteolysis. In
addition, these studies indicated that the inhibition of proteasomal
proteolysis results in an increase in AANAT2 activity. This indication
was examined further in vitro using other proteasomal
proteolysis inhibitors and pike pineal organs, and pineal organs from
two other species, trout and seabream. Treatment with the indicated
inhibitors of proteasomal proteolysis elevated AANAT2 activity 1.5- to
4-fold (Table 1; Fig. 6), suggesting that
low levels of AANAT2 activity during the day appear to reflect in part
continuous degradation by proteasomal proteolysis.
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; Fig. 6). Treatment for 6 h at
this time, with some, but not all, inhibitors, significantly increased
AANAT2 activity in the trout and pike; in other experiments with trout,
stimulatory effects of proteasomal proteolysis inhibitors were apparent
within 3 h of treatment (not shown). Effects of proteasomal
proteolysis inhibitors on AANAT in seabream pineal glands during the
night were not consistently statistically significant (e.g.
Table 2 vs. Fig. 6), reflecting uncontrolled
variability.
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As indicated above, light exposure at night causes a decrease in AANAT2
activity. This was confirmed here: a 30- to 60-min exposure to light
suppressed in vitro activity 50–80% (Fig. 5). This light-induced decrease in AANAT2
activity was prevented or reduced by treatment with proteasomal
proteolysis inhibitors (Fig. 5), which resulted in levels of AANAT2
activity that were equal to, or greater, than nocturnal values (Figs. 5 and 6).
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). Treatment with forskolin and a
proteasomal proteolysis inhibitor in the light, produced a larger
response than that produced by either drug alone, there was, however,
no evidence of marked potentiation (Fig. 6, seabream and pike). In
dark-adapted pineal organs, no further increase was observed. One hour
of light after 6 h of dark resulted in a reduction in AANAT2
activity that was prevented by the cotreatment with forskolin and MG132
but not either drug alone (Fig. 6, pike). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Daily and photic regulation of AANAT2 protein. The indication that day/night and photically regulated changes in AANAT2 activity are associated with changes in AANAT2 protein comes from in vivo and in vitro studies in pike. AANAT2 protein was more abundant during the night than during day. These changes in AANAT2 protein closely parallel changes in AANAT activity reported here and in the literature (12, 13).
The link between protein and activity was also found to persist in the pike following two lighting manipulations, i.e. when animals were exposed to an hour of light during the night, or to an hour of darkness during the day. In both cases there were similar changes in both activity and protein. It should be added that exposure to light during the night did not return pAANAT2 activity or protein levels to those seen during the day; this may reflect a high level of ongoing AANAT2 synthesis, because AANAT2 mRNA is maximally elevated during the night (10). In addition, the failure of exposure to darkness during the day to elevate either AANAT2 protein or activity may reflect low levels of AANAT2 mRNA at this time, which would limit AANAT2 protein synthesis. The daily rhythm in AANAT2 mRNA is regulated by the endogenous pineal clock; and, it is possible that this clock might influence AANAT2 protein through other mechanisms. For example, it might act through a second messenger to regulate AANAT2 protein stability.
The in vivo demonstration that there is a close link between AANAT2 protein and activity was supported by the results of in vitro studies, which consistently found that AANAT2 activity and protein changed in parallel. Thus it appears that an important element regulating AANAT2 activity is the abundance of AANAT protein. However, it should be noted that changes in AANAT2 activity reported here and in the literature typically are not as dramatic as those seen in melatonin synthesis (27). This difference is of special importance because it might reflect regulation of melatonin synthesis at other steps in the conversion of tryptophan to melatonin or in melatonin destruction. For example, tryptophan hydroxylase and melatonin deacetylation have been identified as a potential regulatory point in melatonin synthesis (28, 29). It is also possible that AANAT2 activity as measured in homogenates is not an entirely reliable indicator of the activity of AANAT within the cell, which might be subject to regulation—either inhibition or activation—that is not apparent following homogenization (30). It has also been pointed out that melatonin synthesis may be regulated by negative feedback mechanisms (31, 32).
cAMP regulation of AANAT2. The organ culture studies presented here make it clear that regulatory pathways exist in the fish pineal gland through which cAMP can alter AANAT2 protein. This is consistent with the highly conserved nature of PKA sites in the AANAT family and evidence from mammalian and avian pineal studies, which indicate that AANAT protein is subject to cAMP regulation (12, 13). The mechanism through which cAMP is thought to act involves inhibition of proteasomal proteolysis (13). It seems reasonable to suspect that this process is also functional in the fish pineal, as discussed in the following section. Accordingly, it is reasonable to consider that cAMP can regulate AANAT protein and activity by inhibiting proteasomal proteolysis.
An important issue to address is whether this regulatory pathway is involved in the physiological control of AANAT2. Reliable in vivo physiological data on pineal cAMP from any fish species is not available. Analysis of cAMP in superfused pineal organs and static cultures of fish photoreceptor cells have revealed changes in cAMP consistent with a role in regulating AANAT2 activity and protein (6). However, similar changes are not seen in static organ cultures (15, 33) in which AANAT2 activity or melatonin synthesis did change. This failure to detect changes in cAMP may indicate that other second messenger systems, including calcium, participate in regulating AANAT2 activity and melatonin synthesis. It is also possible that dramatic compartmented changes in cAMP occur in the fish pinealocyte, which are undetectable when the gland is homogenized. Such highly compartmentalized changes, which could occur in discrete regions in which AANAT2 destruction occurs, are not unrealistic to propose, when one considers the organization of the fish pinealocyte and the heterogeneous nature of cells in the pineal organ. The fish pinealocyte is similar to a retinal photoreceptor, and has distinct outer and inner segments, cell body, and pedicles. The fish pineal organ contains photoreceptors, glia, and neurons. Accordingly, cAMP levels in nonmelatonin producing cells might mask dramatic compartmentalized subcellular changes in melatonin-producing cells.
Proteasomal proteolysis of AANAT2. The conclusion that pineal AANAT2 is subject to proteasomal proteolysis in the fish pineal organ is supported by the results of pharmacological studies of three species presented here. In these studies, inhibitors of the proteasomal degradation pathway (34, 35) increased the abundance of pineal AANAT2 activity and protein under a variety of conditions in which AANAT2 protein and activity are otherwise at low levels. For example, inhibition of proteasomal degradation increases AANAT2 activity (pike and seabream) and protein (pike) during subjective night in the light. In addition, treatment with inhibitors of proteasomal degradation at night in culture, inhibits the light-induced decrease in AANAT2 protein and activity. In the case of trout, inhibitors of proteasomal degradation increase AANAT2 activity during the day or subjective day in the dark. This finding indicates that proteasomal proteolysis contributes to the low steady-state levels of AANAT2 protein and activity seen during the day, and that this route of metabolism appears to play a role in the light-induced decrease in these parameters.
Under conditions in which AANAT2 protein and activity are already elevated, including darkness at night, proteasome inhibitors were found to either have no effect in the dark, or to enhance the dark-induced rise in AANAT2 activity. In the case of enhancement, it would appear that proteasomal proteolysis of AANAT2 is ongoing at night, albeit at levels relatively reduced from day levels; in the case were no observed effect of these inhibitors was seen, it would appear that proteasomal proteolysis was maximally inhibited.
We suspect that proteolysis at night is most probably of physiological importance and modulates the amplitude of the AANAT2 peak in the dark, and the shape of the AANAT2 oscillations throughout the LD cycle. Indeed, in species displaying AANAT2 mRNA oscillations (e.g. pike), a basal level of proteolysis at night would cause AANAT2 activity to closely follow changes in AANAT2 mRNA that occur before the dark to light transition (10). Light-activated proteolysis during the day would also prevent AANAT2 activity from increasing dramatically before night onset, when AANAT2 mRNA abundance is increasing. In the case of the trout, where the abundance of AANAT2 mRNA remains constant throughout the LD cycle (18), it would appear that the photically regulated difference in proteasomal proteolysis is the major mechanism driving the daily rhythm in AANAT2 activity and melatonin production (20, 21).
It is relevant to comment on a report that failed to observe an effect of proteasomal inhibitors on AANAT2 activity in the trout pineal organ (15), in contrast to studies presented here, where proteasomal inhibitors were found to statistically increase AANAT2 protein and activity under a variety of conditions. Although the effects of proteasomal inhibitors were seen in all three species examined, their effectiveness varied somewhat on a species-to-species basis. Small effects of the proteasome inhibitors in this species may have occurred, but have been masked by the large variation in AANAT2 activity. Similarly, it is possible that this characteristic of the trout pineal gland might explain the failure of others to observe statistically significant effects of proteasomal proteolysis inhibitors. Other technical differences, including culture conditions (9, 11, 32, 36) and duration of treatment, might also contribute to the differences observed. As discussed above, it is possible that large changes in melatonin production might be due to large changes in the activity of AANAT2 in the intact cell that are not seen when the enzyme is homogenized and AANAT2 is assayed under ideal conditions. For example, protein might be inhibited in the cell during the day, whereas this inhibition is not apparent in broken cells assays. This would explain the small differences between daytime and nighttime AANAT2 values or between daytime values of organs treated with or without proteasome inhibitors.
Conclusions
Three major findings emerge from this study. First, the daily
variations in AANAT2 activity result from variations in the amount of
AANAT2 protein. Second the acute inhibitory effect of light on enzyme
activity results from direct proteolysis of the protein, through
proteasomal proteolysis, independent of the presence or not of a
circadian clock. This receptor-mediated process provides the
photoreceptor cells with a rapid switch to turn off AANAT2. The
magnitude of the turn-on depends on the amount of mRNA present at a
given time of the LD cycle. In clock-controlled pineal organs, the
dark-induced rise in activity will occur only when high mRNA levels
coincide with low proteolysis; but in the absence of clock-controlled
mRNA oscillations AANAT2 activity will increase any time of the 24
h cycle darkness is applied. Third, a cAMP-dependent pathway protects
AANAT2 protein from degradation; this may involve phosphorylation of
AANAT through highly conserved N- and C-terminal cAMP-dependent protein
kinase phosphorylation sites. Proteasomal proteolysis appears as a
conserved mechanism between vertebrate species as divergent as the rat
and the fish are, although the mechanisms through which the process is
triggered are different.
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Footnotes |
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Received August 17, 2000.
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 S. L. Coon, J. L. Weller, H.-W. Korf, M. A. A. Namboodiri, M. Rollag, and D. C. Klein cAMP Regulation of Arylalkylamine N-Acetyltransferase (AANAT, EC 2.3.1.87). A NEW CELL LINE (1E7) PROVIDES EVIDENCE OF INTRACELLULAR AANAT ACTIVATION J. Biol. Chem., June 22, 2001; 276(26): 24097 - 24107. [Abstract] [Full Text] [PDF]
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the mean value.
Day, Pineal organs sampled during day (before the experiment started);
C, control for each lighting condition; F, forskolin
(10-5 M); MG, MG132
(10-4 M). Similar results were
obtained when forskolin was replaced by the nonspecific
phosphodiesterase inhibitor isobutylmethylxanthine (IBMX; not shown).
For further details see Materials and Methods. Similar
effects on AANAT2 activity were obtained in the experiment in Fig. 6
