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The Journal of Neuroscience, July 1, 1998, 18(13):4946-4952
Acetylcholine Triggers L-Glutamate Exocytosis via
Nicotinic Receptors and Inhibits Melatonin Synthesis in Rat
Pinealocytes
Hiroshi
Yamada1,
Akihiko
Ogura2,
Shinichi
Koizumi3,
Akihito
Yamaguchi1, and
Yoshinori
Moriyama1
1 Department of Cell Membrane Biology, Institute of
Scientific and Industrial Research (ISIR), Osaka University, Ibaraki,
Osaka 567, Japan, 2 Department of Biology, Osaka University
Graduate School of Science, Toyonaka, Osaka 560, Japan, and
3 Novartis Pharma K. K., Takarazuka 665, Japan
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ABSTRACT |
Rat pinealocytes, melatonin-secreting endocrine cells, contain
peripheral glutaminergic systems. L-Glutamate is a negative regulator of melatonin synthesis through a metabotropic
receptor-mediated inhibitory cAMP cascade. Previously, we reported that
depolarization of pinealocytes by externally added KCl and activation
of L-type Ca2+ channels resulted in secretion of
L-glutamate by microvesicle exocytosis. What is unknown is
how and what kinds of stimuli trigger glutamate exocytosis under
physiological conditions. Here, we report that the nicotinic
acetylcholine receptor can trigger glutamate exocytosis from cultured
rat pinealocytes. Moreover, acetylcholine or nicotine inhibited
norepinephrine-dependent serotonin N-acetyltransferase activity, which
results in decreased melatonin synthesis. These activities were blocked
by (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine, an antagonist of
the metabotropic glutamate receptor. These results suggest that
cholinergic stimulation initiates the glutaminergic signaling cascade
in pineal glands and that parasympathetic neurons innervating the gland
exert negative control over melatonin synthesis by way of the
glutaminergic systems.
Key words:
microvesicle (synaptic-like microvesicle); acetylcholine; nicotinic acetylcholine receptor (nAchR); exocytosis; glutamate; pinealocyte; pineal gland; L-type Ca2+ channel; serotonin N-acetyltransferase; parasympathetic neuron; melatonin
synthesis
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INTRODUCTION |
The mammalian pineal gland is a
photoneuroendocrine transducer that rhythmically synthesizes and
secretes melatonin at night in response to photoperiodic stimuli and
signals from endogenous circadian oscillators (for review, see Axelrod,
1974 ; Klein, 1985; Reiter, 1991). In the rat, this process is
controlled by sympathetic neurons projecting into the glands. These
neurons secrete norepinephrine (NE), which binds to adrenergic  1 and
 1 receptors. Stimulation of 1 receptors causes increased
intracellular cAMP leading to transcriptional activation of the
serotonin N-acetyltransferase (NAT) gene, which results in increased
melatonin output (for review, see Foulkes et al., 1997). Furthermore,
stimulation of 1 receptors potentiates the activation effect of 1
receptors by increasing intracellular Ca2+
concentration ([Ca2+]i) by release from
intracellular stores (Klein, 1985). On the basis of this information,
the role of sympathetic innervation as a positive regulatory mechanism
for melatonin synthesis has been firmly established.
In addition to adrenergic pathways, there is evidence for other types
of neuronal control of synthesis and secretion of melatonin (Reiter,
1991; Korf et al., 1996). For example, morphological and
immunohistochemical evidence suggests parasympathetic innervation of
mammalian pineal glands (for review, see Moller, 1992; Laitinen et al.,
1995). These neurons may originate from the pterygopalatine ganglion
and use acetylcholine as a neurotransmitter (Moller, 1992; Laitinen et
al., 1995) to activate nicotinic and muscarinic acetylcholine receptors
that have been identified in the pineal gland (Wada et al., 1989;
Pujito et al., 1991a; Reuss et al., 1992; Stankov et al., 1993). After
binding of acetylcholine to receptors, NE-dependent melatonin synthesis
is markedly inhibited, which suggests a negative regulatory role of
parasympathetic innervation (Pujito et al., 1991b; Stankov et al.,
1993; Drijfhout et al., 1996). The mechanism by which acetylcholine
inhibits melatonin synthesis is not known, and the question of whether
muscarinic or nicotinic receptors participate in this inhibitory
processes is still controversial.
Peripheral glutaminergic systems recently identified in pineal glands
are also involved in negative regulation of melatonin synthesis (for
review, see Moriyama et al., 1996). Pinealocytes secrete
L-glutamate through microvesicle-mediated exocytosis
(Yamada et al., 1996a) to affect inhibition of NE-dependent melatonin synthesis. In this case, activation of metabotropic type 3 glutamate receptors (mGluR3) initiates an inhibiting cAMP cascade and results in
decreased NAT activity (Yamada et al., 1998). Under in vitro experimental conditions, glutamate exocytosis can be triggered by the
addition of external KCl, which depolarizes the cell membrane and
activates L-type Ca2+ channels (Yamada et al.,
1996a,b; Yatsushiro et al., 1997). The in vivo stimuli that
initiate glutamate exocytosis remain to be understood.
Recently, Korf and colleagues (Letz et al., 1997) found that
acetylcholine will depolarize pinealocyte membranes by activation of
nicotinic acetylcholine receptor (nAchR). Combining this observation with our recent findings (Yamada et al., 1996b), we hypothesized that
acetylcholine- and glutamate-evoked signaling cascades overlap. Here,
we report that in rat pineal glands acetylcholine triggers glutamate
exocytosis via nAchR and inhibits NE-dependent melatonin synthesis
through an inhibitory cAMP cascade. Our results suggest that
parasympathetic neurons negatively control melatonin synthesis by way
of endogenous glutaminergic systems in pineal glands.
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MATERIALS AND METHODS |
Cell culture. Cells were isolated from the pineal
glands of Wistar rats at 7 weeks postnatal and were cultured by
published procedures (Yamada et al., 1996a,b). Briefly, pineal glands
were dissected into small pieces, treated with a 0.1% collagenase
solution (Life Technologies, Gaithersburg, MD) at 37°C for 30 min
with gentle shaking, and then washed with PBS. After centrifugation at
180 × g for 2 min, the pieces were treated with a
0.025% trypsin solution (Life Technologies) at 37°C for 20 min and
then centrifuged at 180 × g for 5 min. The dispersed
cells were washed three times with DMEM supplemented with 6% fetal
calf serum, 55 mg/ml sodium pyruvate, 6 mg/ml glucose, 0.1 mg/ml
streptomycin, 100 U/ml penicillin G, and 0.25 mg/l fungizone and placed
in a culture dish (35-mm-diameter) coated with type I collagen
(Corning, Corning, NY) at 1.3 × 106
cells/dish. Cells were cultured in the above medium at 37°C under 10% CO2. More than 99.5% cells that became attached to
the dish were pinealocytes, as shown by the positive reactivity toward anti-melatonin and anti-synaptophysin antibodies (Yamada et al., 1996a). The viability of the cells did not change after treatment with
the various reagents used in this study, as judged by staining with
0.3% Trypan blue and vital staining with acridine orange.
Assay of glutamate exocytosis. Pinealocytes (1.3 × 106 cells/dish) were preincubated at 28°C for 30 min and then washed with Ringer's solution composed of 128 mM NaCl, 1.9 mM KCl, 1.2 mM KH2PO4, 2.4 mM
CaCl2, 1.3 mM MgSO4,
26 mM NaHCO3, 10 mM glucose, and 10 mM HEPES, pH 7.4, or
 Ca2+-Ringer's solution composed of 128 mM NaCl, 1.9 mM KCl, 1.2 mM KH2PO4, 0.2 mM
CaCl2, 1 mM EGTA, 3.8 mM
MgSO4, 26 mM NaHCO3,
10 mM glucose, and 10 mM HEPES, pH 7.4. After
cells were incubated in 2 ml of the above medium at 28°C, exocytosis
was stimulated by the addition of either 50 mM KCl (Yamada
et al., 1996a) or 0.2 mM acetylcholine. When necessary,
various Ca2+ channel agonists or antagonists were
included in the incubation medium. Finally, 10 µl aliquots were taken
at intervals, and the amount of extracellular glutamate was determined
by HPLC with precolumn o-phthalaldehyde (OPA) derivatization,
separation by a reverse-phase RESOLVE C18 column (3.9 × 150 mm;
Waters Associates, Milford, MA), and fluorescence detection (Godel et
al., 1984).
Treatment of pinealocytes with botulinum neurotoxin type E
(BoNT/E). Pinealocytes were treated with BoNT/E by a
procedure similar to that described previously (Yamada et al., 1996a;
Yatsushiro et al., 1997). In brief, pinealocytes (1.3 × 106 cells/dish) were incubated at 37°C for 24 hr
in a low ionic strength buffer consisting of 5 mM NaCl, 4.8 mM KCl, 2.2 mM CaCl2, 1.2 mM MgSO4, 20 mM HEPES-NaOH,
pH 7.4, 5.6 mM glucose, 220 mM sucrose, and
0.5% bovine serum albumin in the presence or absence of either 15 nM or 50 nM BoNT/E. After treatment, cells were
washed with fresh culture medium and incubated an additional 12 hr at
37°C. Finally, acetylcholine-evoked glutamate release was measured as described above.
Measurement of intracellular [Ca2+].
Pinealocytes were cultured for 3 d on a thin glass coverslip
precoated with type I collagen (thickness 0.12 mm and diameter 40 mm;
8.0 × 105 cells/coverslip) in the medium as
described (Ogura et al., 1990). After changing the culture medium to
DMEM, cells were treated with 5 µM fura-2 AM (Dojin
Company) for 50 min at 37°C in DMEM and then washed twice with the
same medium. The cells were perfused with warmed Ringer's solution or
Ca2+-Ringer's solution, and images were
continuously taken at 28°C with a silicon-intensified target camera
(Hamamatsu Photonics C-2400-08) at a sampling rate of 30 sec-1, and recorded on videotape. The output of the
camera or of the videotape recorder (Sony V0-5850) was fed into a
Hamamatsu DVS-3000 image analyzer. The software enabled subtraction of
background fluorescence, pixel-to-pixel division of F340 × F360
images, fitting of the F340/F360 ratios to a
[Ca2+] calibration curve prepared separately, and
digital averaging of [Ca2+] in multiple cells.
Other procedures. Melatonin was measured by HPLC with an
IRICA RP-18T column and an amperometric detector (E-558) as described by Sagara et al. (1988). NAT activity was measured as described by
Thomas et al. (1990). Immunoblotting and PAGE were performed as
described previously (Moriyama and Yamamoto, 1995a,b). cAMP concentrations were measured with an Amersham cAMP enzyme-immunoassay kit (Amersham, Arlington Heights, IL), and protein was measured by the
Bio-Rad protein assay kit (Bio-Rad, Richmond, CA).
Materials. Antagonists and agonists of acetylcholine
receptor were from Sigma (St. Louis, MO).
1,4-Dihydro-2,6-dimethyl-5-nitro-4[trifluoromethyl-phenyl]3-pyridinecarboxylic acid methylester (BAY K8644) was obtained from Research Biochemicals International (Natick, MA).
(2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), a specific
antagonist for class II mGluRs (Sekiyama et al., 1996), was obtained
from Tocris Neuramin. BoNT/E was kindly provided by Professor S. Kozaki
(Osaka Prefecture University). Monoclonal antibodies against synaptic
vesicle-associated protein 25 (SNAP25) (BR05) and polyclonal antibodies
against synaptobrevin 2 (VAMP2) were obtained from Wako Chemicals
(Osaka, Japan). Other chemicals were of the highest grade commercially
available.
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RESULTS |
Acetylcholine-evoked L-glutamate exocytosis
Our previous studies indicated that L-glutamate in
microvesicles was exocytized from pinealocytes after depolarization of the cell membrane evoked by the addition of 50 mM or more
KCl (Yamada et al., 1996a). A rise of intracellular
[Ca2+] via voltage-gated L-type
Ca2+ channels is responsible for the secretion
process (Yamada et al., 1996b). We report here that acetylcholine can
also stimulate appreciable amounts of L-glutamate
exocytosis (Fig. 1A).
The rate of secretion increases in a saturable manner with increasing
concentrations of acetylcholine with a calculated Km value of 63 µM (Fig. 1B). Approximately 1.8 nmol of
glutamate was released from 106 cells after a 10 min
incubation with acetylcholine. Acetylcholine-induced glutamate
secretion was dependent on temperature: release was observed at 28°C,
decreased gradually with decreasing temperature, and disappeared below
18°C (Fig. 1A). Above 32°C, pinealocytes became
sensitive to mechanical stimulation such as medium replacement and
readily released glutamate even in the absence of acetylcholine. At
28°C, essentially no glutamate was released by mechanical stimulation such as medium replacement. Because of this behavior, subsequent experiments were performed at 28°C to ensure quantitative secretion measurements. Involvement of either eserine or neostigmine at 50 µM, inhibitors of acetylcholine esterase, did not affect
the acetylcholine-induced glutamate secretion. This excludes a
possibility that the enzyme perturbed the acetylcholine effect under
the assay condition. After a round of glutamate release, successive
additions of acetylcholine did not induce further secretion. The
ability to secrete glutamate gradually returned during a 12 hr period (data not shown). These properties were consistent with those of
KCl-evoked glutamate secretion (Yamada et al., 1996a), suggesting that
acetylcholine-evoked glutamate secretion is also mediated through
microvesicle exocytosis.
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Figure 1.
Characterization of acetylcholine-evoked
L-glutamate secretion from cultured pinealocytes.
A, Glutamate concentration in the medium at the
indicated times after the addition of 200 µM
acetylcholine was monitored as described in Materials and
Methods. Cells were incubated at 28°C ( ), 22°C ( ), or 18°C
( ). Results were expressed as mean ± SEM (4 independent
experiments). B, Lineweaver-Burk plot of acetylcholine
dose-dependence after 10 min. The derived Km
and Vmax values were 63 µM and
0.22 nmol/min per 106 cells, respectively.
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We conducted two lines of experiments to show that acetylcholine-evoked
exocytosis involved microvesicles. First, we assessed the role of
Ca2+ (Fig. 2). Removal
of Ca2+ from the medium reduced acetylcholine-evoked
glutamate secretion by 80%. Diltiazem, nifedipine, and nitrendipine,
L-type Ca2+ channel blockers (Tsien et al., 1988),
inhibited ~50% of acetylcholine-evoked glutamate release, whereas
BAY K8644, an L-type Ca2+ channel agonist (Bellemann
and Frankowiak, 1985; Nowycky et al., 1985), stimulated secretion
twofold over the acetylcholine response. These results strongly suggest
the involvement of L-type Ca2+ channels in the
secretion mechanism.
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Figure 2.
Involvement of L-type Ca2+
channels in acetylcholine-evoked glutamate secretion. Cells were
treated with the indicated L-type Ca2+ channel
antagonists (20 µM each) or BAY K8644 (1 µM) for 10 min and washed once with medium. Cells were
then stimulated with 200 µM acetylcholine for 10 min, and
aliquots of medium were taken for determination of glutamate. The
results were expressed as relative values ± SEM (three
independent experiments). The amount of glutamate released on addition
of 5 µM Ca2+ ionophore A23187 (2.2 nmol glutamate/106 cells) was taken as 100%, and
glutamate secretion in the absence of Ca2+ in the
medium (Ca2+-Ringer's solution) is shown to
indicate background levels (control levels).
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Second, we assessed participation of SNAP25, a synaptic vesicle protein
that is a component of the SNARE (SNAP receptor) complex (Damer and
Creutz, 1994; Rothman, 1994; Scheller, 1995; Südhof, 1995).
Botulinum toxin cleaves SNAP25 and interferes with microvesicle docking
and fusion during the exocytotic process (Yamada et al., 1996b;
Yatsushiro et al., 1997). Indeed, immunoblot analysis using SNAP25
antibodies shows that BoNT/E treatment of pinealocytes resulted in
cleaved SNAP25 without affecting VAMP2 (Fig.
3, top panel).
Significantly, BoNT/E treatment also resulted in inhibited acetylcholine-evoked glutamate secretion in a toxin
concentration-dependent manner (Fig. 3). Together with the results
indicating the role of Ca2+, these results are fully
consistent with the properties of microvesicle exocytosis (Yamada et
al., 1996a,b) and strongly suggest that acetylcholine triggers
glutamate secretion through this process.
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Figure 3.
Effects of BoNT/E on acetylcholine-evoked
glutamate secretion. Cells were treated with BoNT/E at the indicated
concentrations as described in Materials and Methods. After treatment,
acetylcholine-evoked glutamate release for 10 min was determined.
Results in the bottom panel are expressed as relative
values ± SEM (4 independent experiments); 100% value is 1.8 nmol
glutamate/106 cells. In the top
panel, the same cells were washed with PBS containing DNase and
dissolved in sample buffer containing 10% SDS and -mercaptoethanol.
After dissociation of the proteins, samples were separated over a 13%
polyacrylamide gel in the presence of SDS and analyzed by
immunoblotting using antibodies against SNAP-25 and VAMP2 as indicated.
The position of cleaved SNAP-25 is indicated by *.
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Involvement of nAchR in glutamate secretion
Acetylcholine binds to both nicotinic and muscarinic acetylcholine
receptors. To determine which type of receptor is involved in
pinealocyte glutamate secretion, pharmacological analysis was conducted. As shown in Figure
4A, glutamate secretion
was triggered by the addition of nicotine but not muscarine: the
Km and Vmax values for
nicotine were 77 µM and 0.21 nmol/min per
106 cells, respectively (Fig. 4B).
Both nicotine- and acetylcholine-evoked glutamate secretion were
inhibited 80% by the addition of tubocurarine, a competitive inhibitor
of the nAchR, whereas scopolamine and atropine, antagonists of the
muscarinic receptor (Pujito et al., 1991b), or -bungarotoxin, a
selective inhibitor for some subtypes of nAchRs, were ineffective (Fig.
4A). Carbachol at 0.2 mM, an agonist for
both muscarinic and nicotinic receptors (Pujito et al., 1991b),
stimulated secretion. Neither scopolamine nor atropine at 400 µM affected the carbachol-induced glutamate secretion
(data not shown). These results specify that an
-bungarotoxin-insensitive nAchR is responsible for glutamate
secretion.
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Figure 4.
Pharmacological (A) and
nicotine () and muscarine () dose-dependent
(B) effects on glutamate secretion in the
presence or absence of acetylcholine receptor antagonists at 10 min
were determined as described in the legend of Figure 1. Results are
expressed as relative values (A) or means
(B) ± SEM (4 independent experiments); 100%
activity is 1.7 nmol glutamate/106 cells. Reagents
at the following concentrations were used: acetylcholine and receptor
agonists, 200 µM; tubocurarine, scopolamine and atropine,
400 µM; and -bungarotoxin, 100 nM.
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The requirement for extracellular Na+ will also
distinguish nicotinic and muscarinic receptors. Replacement of
Na+ with the nonpermeating cation
N-methyl-D-glucamine (NMDG) (Letz et al., 1997)
significantly decreased acetylcholine-evoked glutamate secretion (Fig.
5). The same pinealocytes exhibited
normal levels of KCl-evoked glutamate secretion, indicating that the
cells retain microvesicle exocytosis capability. Because
Na+ is required for nAchR function (see below),
these results further supported the involvement of nAchR but not
muscarinic receptor in acetylcholine-evoked glutamate secretion.
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Figure 5.
Na+ requirement for
acetylcholine-evoked glutamate secretion. Cells were incubated in
Ringer's solution (Ringer) or Ringer's solution
containing NMDG (NMDG). The acetylcholine-evoked
glutamate released at 10 min was determined. In NMDG + KCl, 50 mM KCl was added in Ringer's solution
containing NMDG to initiate glutamate secretion. Results are expressed
as relative values ± SEM (4 independent experiments); 100%
activity is 1.8 nmol glutamate/106 cells.
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Functional coupling between nAchR and L-type
Ca2+ channel
After activation, the nAChR channel increases
Na+ conductance and depolarizes the pinealocyte
membrane (Letz et al., 1997). Depolarization activates L-type
Ca2+ channels, and the resultant increase of
intracellular [Ca2+] should trigger microvesicle
exocytosis. To demonstrate that the above cascade is operating in our
experimental conditions, we observed acetylcholine- or nicotine-evoked
intracellular [Ca2+] transients in fura-2
AM-loaded pinealocytes in the presence or absence of various channel
blockers. Either acetylcholine or nicotine evoked increased
intracellular [Ca2+] in pinealocytes in a
dose-dependent manner (Fig.
6A-C). Apparent Km values for acetylcholine and nicotine are 86 and 90 µM, respectively (Fig. 6B). A
rise of intracellular [Ca2+] required
extracellular Ca2+ and was inhibited by L-type
Ca2+ channel blockers such as nifedipine (data not
shown). Removing Na+ from the medium also blocked
the acetylcholine effect (data not shown), as did tubocurarine (Fig.
6D). Although carbachol stimulated increased
intracellular [Ca2+], no muscarinic acetylcholine
receptor agonists caused increased intracellular
[Ca2+] (Fig. 6E). Essentially
the same effects on the requirement of extracellular
Na+ and Ca2+ and on inhibitor
sensitivities were observed in the case of nicotine-evoked [Ca2+] transients (data not shown). These results
were consistent with the previous observations by Letz et al. (1997).
We conclude that nAchR and L-type Ca2+ channel are
functionally coupled in pinealocytes.
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Figure 6.
Acetylcholine or nicotine increases intracellular
[Ca2+] in pinealocytes. A, Typical
intracellular [Ca2+] transients as monitored by
fura-2 AM fluorescence. A fluorescence micrograph was shown to indicate
fura-2 AM-loaded pinealocytes (left). At 0 time,
acetylcholine (200 µM) was added and the change in fura-2
AM fluorescence within individual pinealocytes was followed.
B, Dose dependencies of acetylcholine () and nicotine
() are shown. Results are expressed as means of peak values at ~15
sec ± SEM obtained from 20 cells. C, The majority
of pinealocytes respond after the addition of acetylcholine or nicotine
(200 µM each). D, E, Traces of fura-2 AM
fluorescence indicating intracellular [Ca2+]
transients from a single pinealocyte. The following compounds were
added as indicated by the bars: 200 µM
acetylcholine, 200 µM nicotine, 1 µM NE,
400 µM tubocurarine, 200 µM muscarine, 200 µM carbachol, or 200 µM oxotremorine.
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Acetylcholine-evoked inhibition of melatonin synthesis
We showed previously that L-glutamate secreted from
pinealocytes may inhibit NE-dependent melatonin synthesis via the
mGluR3-mediated inhibitory cAMP cascade (Yamada et al., 1998). As a
final step of the study, we tested whether stimulation of nAchR would
result in inhibition of NE-dependent melatonin synthesis as well.
As expected, either acetylcholine or nicotine strongly inhibited
NE-dependent melatonin synthesis with 50% inhibition attained at 75 and 60 µM, respectively (Table
1). Muscarine at 0.2 mM had
only a slight effect. The nicotine-evoked inhibition was prevented by
the treatment of 0.4 mM tubocurarine. Consistent with the
glutaminergic signaling cascade, either acetylcholine or nicotine also
inhibited NE-dependent NAT activity (Table 1). Inhibition of melatonin synthesis and NAT activity were recovered to control levels after treatment with 2 mM dibutylyl cAMP, a nonhydrolyzable cAMP
analog (data not shown). Furthermore, the acetylcholine- or
nicotine-evoked inhibition was blocked when pineal glands were treated
with MCCG, a specific antagonist of class II mGluR. This compound also
blocked glutamate action in the pineal gland (Yamada et al., 1998)
(Table 1). These results clearly showed that the class II
mGluR-mediated inhibitory cAMP cascade is involved in
acetylcholine-evoked inhibition of melatonin synthesis.
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DISCUSSION |
In this study, we report that acetylcholine triggers glutamate
exocytosis from rat pinealocytes by way of nAchR and L-type Ca2+ channels. The data presented here and in
previous reports are consistent with the following signaling cascade:
activation of nAchR depolarizes the pinealocyte membrane by increasing
Na+ conductance. Depolarization activates L-type
Ca2+ channels resulting in increased intracellular
[Ca2+], which in turn triggers microvesicle
exocytosis. Secreted glutamate activates mGluR3 receptors leading to
inhibited NE-dependent melatonin synthesis (Yamada et al., 1998).
Although the physiological significance of nAchR has yet to be
established, the results reported here clearly indicate functional
coupling between nAchR and L-type Ca2+ channels and
participation in the glutaminergic inhibitory mechanism of melatonin
synthesis in pineal glands.
To our knowledge, this is the first example of nAchR regulation of
glutamate secretion in endocrine cells. Enhancement of neurotransmitter
release on stimulation of nAchR has been observed in various neurons.
nAchRs are classified into two categories based on sensitivity to
nicotine and -bungarotoxin (for review, see Role and Berg, 1996).
One class requires relatively high concentrations of nicotine (~100
µM) for activation and is insensitive to
-bungarotoxin, whereas the other type activates at low nicotine
concentrations (<1 µM) and is sensitive to
-bungarotoxin. Glutamate secretion in pinealocytes seems to be of
the former type, because a high concentration of nicotine was required
for activation and the receptor was insensitive to -bungarotoxin
(Fig. 4). This is consistent with in situ hybridization
studies reporting the presence of -bungarotoxin-insensitive 32
subunits in the pineal gland (Wada et al., 1989).
Recently, Pujito et al. (1991b) and Drijfhout et al. (1996) reported
that muscarinic receptor agonists strongly inhibited melatonin
synthesis in rat pineal gland; however, such inhibition was not
observed in our hands (Table 1). Muscarinic receptor agonists appear to
target presynaptic membranes and block NE release from sympathetic
neurons. This effect causes decreased melatonin output (Drijfhout et
al., 1996). Such muscarinic agonist action would not be observed in our
assay conditions because exogenous NE was included to ensure full
melatonin output. It is reasonable that acetylcholine has alternative
pathways to inhibit NE-dependent melatonin synthesis. One pathway
involves presynaptic modulation of NE release through muscarinic
acetylcholine receptors (Drijfhout et al., 1996) and the other involves
nAchR-mediated glutamate-evoked inhibitory cascade as shown here in
pinealocytes.
We showed that cholinergic and glutaminergic signaling pathways overlap
on the way to the end point of decreased melatonin output. Our
conclusion immediately raises the fascinating hypothesis that the CNS
is responsible for negative regulation of melatonin synthesis. Either
parasympathetic neurons innervating the gland or cholinergic
interneurons present in the gland (Pujito et al., 1991a; Wessler et
al., 1997) may participate in regulation of the melatonin synthesis.
Clearly, more extensive studies are necessary.
In summary, we presented evidence that acetylcholine initiates an
intrinsic glutaminergic system via nAchR to inhibit melatonin synthesis
in pineal glands. This finding may define the major physiological role
of nicotinic acetylcholine receptors in the endocrine system.
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FOOTNOTES |
Received Feb. 27, 1998; revised April 16, 1998; accepted April 20, 1998.
H.Y. is supported by a research fellowship from the Japan Society for
Promotion of Science. This study was supported in part by grants from
the Japanese Ministry of Education, Science and Culture. We thank
Professor H.-W. Korf for valuable discussion, Professor R. K. Nakamoto (University of Virginia) for critical reading of this
manuscript, and Professor S. Kozaki for providing BoNT/E.
Correspondence should be addressed to Dr. Y. Moriyama, Department of
Cell Membrane Biology, Institute of Scientific and Industrial Research
(ISIR), Osaka University, Ibaraki, Osaka 567, Japan.
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Abstract
Introduction
