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INTRODUCTION |
Nitric oxide is a major endogenous mediator involved in many
physiological and pathological functions such as vasodilation, neurotransmission, and cytotoxicity (1-3). In brain, NO is synthesized mainly by nNOS1 (4), which is
expressed in various brain regions including the cerebellum, olfactory
bulb, and several hypothalamic nuclei (5). One of the nNOS-positive
nuclei in the hypothalamus is the suprachiasmatic nucleus (SCN) (6),
which has a circadian oscillator to create circadian rhythms in
hormonal secretions, enzyme activities, and behaviors. The SCN also
controls energy metabolism through the regulation of the autonomic
nervous system (7). We have previously shown that
NG-methylarginine, an inhibitor of NOS, disturbs
the circadian rhythm of drinking behavior in rats, suggesting that NO
is involved in the generation and/or synchronization of the
circadian rhythm (8).
nNOS is one of three known isoforms of nitric-oxide synthase. Although
nNOS does not have a transmembrane domain, subcellular fractionation
experiments showed that ~60% of the total NOS activity in brain was
found in the particulate fraction, suggesting that nNOS is associated
with membranes by interacting with some other membrane proteins (9).
The N-terminal domain of nNOS is unique to this isoform, having a PDZ
motif, which is found in various structural proteins (10). This domain
of nNOS is reported to interact with PDZ motifs in PSD
(postsynaptic density)-95 and PSD-93 (11) to form macromolecular signaling complexes at postsynaptic sites and possibly to modulate synaptic transmission.
nNOS is expressed not only in neuronal cells, but also in several other
tissues such as the fast-twitch fibers of skeletal muscle (12). In
skeletal muscle, nNOS is targeted to sarcolemmal membranes by
association with another PDZ-containing protein,
1-syntrophin,
through PDZ-PDZ interactions (11). The syntrophins are a multigene
family of proteins including
1,
1, and
2 isoforms, each of
which has one PDZ domain and three pleckstrin domains (13). In
mammalian skeletal muscle, syntrophins are components of the dystrophin
complex at sarcolemmal membranes, and are thought to function as
adaptors that recruit signaling proteins to the membranes (14).
1-Syntrophin is also expressed in brain (15). However, the
interaction of nNOS with
1-syntrophin in brain has not been
precisely investigated yet.
To examine whether the PDZ domain of nNOS interacts with proteins other
than PSD-95 in brain, we purified nNOS-interacting proteins from bovine
brain lysate. In this report, we show that one of the nNOS-interacting
proteins in brain is
1-syntrophin. We also investigated the
localization of nNOS and
1-syntrophin in primary cultured neurons
from rat brain and in neurons from the hypothalamus to gain insight
into the physiological functions of nNOS and nNOS-associated proteins
in the central regulation of metabolism.
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EXPERIMENTAL PROCEDURES |
GST Fusion Proteins--
GST-
1-syntrophin-(31-90),
GST-
1-syntrophin-(69-201), and GST-nNOS-(1-230) constructs
were generated by cloning corresponding sequences, which were amplified
by reverse transcription-polymerase chain reaction from rat brain
cDNA, and introduced into the pGEX vector (Amersham Pharmacia
Biotech, Uppsala, Sweden). GST fusion proteins were expressed in
Escherichia coli and affinity-purified on
glutathione-Sepharose (Amersham Pharmacia Biotech) according to the
manufacturer's protocol.
Antibodies--
Anti-
1-syntrophin polyclonal antibody was
raised in a rabbit against GST-
1-syntrophin-(31-90) fusion protein
and was affinity-purified on a CH-Sepharose column (Amersham Pharmacia
Biotech) coupled with GST-
1-syntrophin. For Western blotting and
immunohistochemistry, this antibody was diluted in Tween/TBS (0.1%
Tween-20, 137 mM NaCl, 2.7 mM KCl, and 10 mM Tris-HCl, pH 7.4) containing 25 µg/ml GST. For
absorption experiments, the antibody was preincubated with
GST-
1-syntrophin-(31-90) or GST-nNOS-(84-230) for 2 h and directly applied to Western blotting, immunocytochemistry, and immunohistochemistry. Anti-nNOS monoclonal antibody targeted to the
N-terminal region of rat nNOS (amino acids 1-180) and anti-GST polyclonal antibody were purchased from Sigma. Anti-nNOS polyclonal antibody targeted to the carboxyl-terminal region of rat nNOS was
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Anti-synaptotagmin monoclonal antibody was purchased from Wako Pure
Chemicals, Co. (Osaka, Japan). Anti-PSD-95 polyclonal antibody was
provided by Dr. T. Akiyama (Institute of Molecular and Cellular
Bioscience, Tokyo University) (16).
Purification and Microsequencing of an nNOS-interacting
Protein--
Purifications of nNOS-interacting proteins were carried
out at 0-4 °C. Bovine brain (500 g) was homogenized in 5 volumes
(w/v) of TNE buffer (25 mM Tris-HCl, 1% Nonidet P-40, and
1 mM EDTA, pH 7.4) containing 0.5 M NaCl and
centrifuged at 15,000 × g for 30 min. The supernatant
was loaded on a GST or GST-nNOS fusion protein column. Each column was
washed with 20 volumes of TNE buffer containing 0.5 M NaCl,
and bound proteins were eluted by addition of 10 mM
glutathione. Eluted proteins were separated by SDS-polyacrylamide gel
electrophoresis (PAGE), electroblotted onto a polyvinylidene difluoride
membrane, and stained with Ponceau S. An nNOS-interacting protein was
digested with a lysylendopeptidase, Achromobacter protease
I, and the fragments yielded were separated by reversed-phase HPLC
(16). Sequences of three of the fragments (AP-1, AP-2, and AP-3) were
determined by peptide microsequencing (17).
Tissue Extraction and Western Blot Analysis--
Rat brains (3.0 g) were homogenized in 10 volumes (w/v) of TNE buffer containing 150 mM NaCl and centrifuged at 15,000 × g for
30 min. The supernatant was resolved by SDS-PAGE and transferred to
nitrocellulose membranes. The membranes were blocked overnight with
Tween/TBS and incubated with primary antibody and then with horseradish
peroxidase-conjugated anti-rabbit IgG (Zymed Laboratories, Inc., South San Francisco, CA). The resultant immunoreactive
bands were visualized by enhanced chemiluminescence
(Renaissance®, NEN Life Science Products) according to the
specifications of the manufacturer.
Protein Overlay Assays--
Rat brain extracts were separated by
SDS-PAGE using 7.5% SDS-polyacrylamide gels and transferred to
nitrocellulose membranes, which were then blocked with Tween/TBS and
incubated with purified GST-
1-syntrophin-(69-201) fusion protein in
Tween/TBS for 1 h at 25 °C. After washing with Tween/TBS, the
blots were incubated with anti-GST antibody and then with horseradish
peroxidase-conjugated anti-rabbit IgG. Bands were visualized by
enhanced chemiluminescence (Renaissance®).
Immunoprecipitations--
Rat brain extracts were pretreated
with protein G-Sepharose (Amersham Pharmacia Biotech) for 1 h at
4 °C. After centrifugation, the supernatants were incubated for
1 h at 4 °C with protein G-Sepharose that was preincubated with
anti-nNOS monoclonal antibody. The Sepharose beads were washed five
times with TNE buffer containing 150 mM NaCl. The resultant
immunoprecipitated proteins were resolved by SDS-PAGE.
Pull-down Assays--
Rat brain extracts (2.5 mg) were incubated
with glutathione-Sepharose beads coupled with GST-nNOS-(1-230) or GST.
The beads were washed three times with TNE buffer containing 0.15 M NaCl and eluted with glutathione (10 mM).
Eluted proteins were analyzed by Western blotting.
Cell Culture and Immunocytochemistry--
Neuronal cultures were
prepared from embryonic day 18 Wistar rats by the method of Brewer
et al. (18) with slight modifications. Briefly, neurons were
isolated by trypsin treatment and plated on
poly-D-lysine-coated glass coverslips in Dulbecco's
modified minimal essential medium with 10% calf serum at a density of
2000 cells/cm2. After attachment of cells, the coverslips
were transferred to serum-free Neurobasal medium (Life Technologies,
Inc.) with N2 supplements. For immunocytochemical staining, the neurons
were fixed with 4% paraformaldehyde 10 days after plating, blocked with 10% bovine serum albumin in Tween/TBS, and exposed to primary antibodies. Primary antibodies were visualized with fluorescein isothiocyanate-conjugated anti-rabbit IgG (ICN Pharmaceuticals, Inc.,
Costa Mesa, CA) or Texas Red-conjugated anti-mouse IgG (Amersham Pharmacia Biotech), which were incubated together for double-labeling experiments. Fluorescent photomicroscopy was performed on a Nikon Diaphot-TMD microscope with Fuji Provia 1600 film.
Immunohistochemistry--
Rats were anesthetized with sodium
pentobarbital and perfused with 4% paraformaldehyde-containing
phosphate-buffered saline. The brains were removed, post-fixed at
4 °C for 2 days, cryoprotected in 30% sucrose at 4 °C for 5 days, and cut into 20-µm sections with a microslicer. The sections
were treated for 3 h in phosphate-buffered saline containing 3%
bovine serum albumin and 0.3% Triton X-100; incubated overnight with
primary antibodies to
1-syntrophin or nNOS, which were diluted into
Tween/TBS; and then incubated for 1 h with horseradish
peroxidase-conjugated anti-rabbit IgG). Signals were visualized with
3,3'-diaminobenzidine (Dojindo Laboratories, Kumamoto, Japan). For
confocal microscopy, signals were visualized with fluorescein
isothiocyanate-labeled anti-mouse IgG and rhodamine-labeled anti-rabbit
IgG and were then observed on a Bio-Rad Micro Radiance confocal
scanning system.
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RESULTS |
Purification and Identification of an nNOS-interacting
Protein--
To examine whether the PDZ domain of nNOS in brain
interacts with proteins other than PSD-95 and PSD-93, we purified
nNOS-interacting proteins by affinity chromatography using
glutathione-Sepharose beads coupled with GST-nNOS-(1-230) fusion
protein. We also used glutathione-Sepharose coupled with GST as a
control. Crude extracts from bovine brain were loaded on each column,
and proteins were eluted with glutathione. We found a protein of ~60
kDa that was eluted from the GST-nNOS affinity column, but not from the
GST column, indicating that this protein was selectively bound to the
N-terminal region of nNOS (Fig.
1A). Because the eluate
contained a large amount of GST-nNOS fusion protein (55 kDa) and its
proteolytic products, proteins with molecular masses <55 kDa were not
analyzed.

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Fig. 1.
Purification and sequence analysis of
nNOS-interacting proteins. A, purification of an
nNOS-interacting protein by GST-nNOS affinity column chromatography.
Crude brain extract was loaded onto a glutathione-Sepharose column
previously coupled with GST or GST-nNOS-(1-230). Bound proteins
together with GST or GST-nNOS were eluted with glutathione, subjected
to SDS-PAGE, and detected by silver staining. The asterisk
denotes an nNOS-interacting protein. B, sequence analysis of
the nNOS-interacting protein. The nNOS-interacting protein was
transferred to a polyvinylidene difluoride membrane and digested with
Achromobacter protease I, and the fragments were obtained by
reversed-phase HPLC. Sequences of three of the fragments(AP-1,
AP-2, and AP-3) were determined by peptide microsequencing and aligned
with those of 1-syntrophin deduced from its cDNA sequence.
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To identify the protein, it was subjected to amino acid sequencing
(Fig. 1B). The protein was digested with
Achromobacter protease I on a polyvinylidene difluoride
membrane, and three peptide fragments (AP-1, AP-2, and AP-3) were
analyzed on a peptide sequencer. A homology search analysis showed that
all these sequences were identical to the fragments of Mus
musculus
1-syntrophin except for the fifth amino acid in the
peptide AP-3 (Fig. 1B). The molecular mass of
1-syntrophin calculated from its cDNA sequence was 58 kDa, which
was close to that of the nNOS-interacting protein estimated by
SDS-PAGE. We therefore concluded that the nNOS-interacting protein was
bovine
1-syntrophin.
Interaction of nNOS with
1-Syntrophin in Brain--
To confirm
whether nNOS can interact with
1-syntrophin, we raised an antibody
against
1-syntrophin-(31-90). We chose this region as an antigen
because it has <50% sequence homology to two other isoforms,
1 and
2. The antibody was affinity-purified with CH-Sepharose coupled with
GST-
1-syntrophin-(31-90). Immunoblotting of a crude rat brain
extract with the antibody showed that it specifically reacted with a
60-kDa protein (Fig. 2A,
lane 1). Some other bands under 45 kDa were also detected,
but they seemed to be nonspecific signals of the secondary antibody
used because they were detected even when the primary antibody was
omitted (Fig. 2A, lane 2).

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Fig. 2.
Interaction of nNOS with
1-syntrophin. A, characterization
of anti- 1-syntrophin antibody. An antibody was raised against
GST- 1-syntrophin-(31-90) in a rabbit and applied to Western
blotting of a crude brain extract (lane 1). As a
control, the extract was treated in the same manner without the primary
antibody (lane 2). The asterisk
denotes 1-syntrophin. B, analysis by pull-down assay.
Crude brain extract was incubated with glutathione-Sepharose coupled
with GST-nNOS-(1-230) fusion protein. After washing the beads, bound
proteins were subjected to SDS-PAGE and analyzed by Western blotting
with anti- 1-syntrophin antibody (lane 3). In
the same gel, purified GST (lane 2) and
GST- 1-syntrophin-(31-90) fusion protein (lane
1) were run to confirm the specificity of the antibody. The
same series of samples was also subjected to Western blotting with
anti- 1-syntrophin antibody preabsorbed with its antigen
(lanes 4-6).
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The specificity of the antibody was further confirmed in Fig.
2B. The antibody reacted with GST-
1-syntrophin (Fig.
2B, lane 1), but not with GST
(lane 2). In addition, binding of the antibody to
GST-
1-syntrophin was completely abolished when it was preincubated with the antigen (Fig. 2B, compare lanes 1 and
4). These results suggest that the antibody specifically
reacts with
1-syntrophin.
Next, we analyzed the interaction of nNOS with
1-syntrophin in brain
by pull-down assay. Glutathione-Sepharose beads coupled with
GST-nNOS-(1-230) were incubated with a rat brain lysate and then
precipitated. Western blotting with anti-
1-syntrophin antibody showed that
1-syntrophin was retained by GST-nNOS-conjugated Sepharose (Fig. 2B, lane 3), but not by
GST-Sepharose (data not shown). The 60-kDa band was not detected when
the antibody was preabsorbed with antigen (Fig. 2B,
lane 6). These results suggest that nNOS
interacts with
1-syntrophin in rat brain.
Analysis of the Interaction between nNOS and
1-Syntrophin by
Overlay Assay--
To confirm the interaction of nNOS with
1-syntrophin and to examine whether binding of nNOS to
1-syntrophin is direct or indirect, we performed protein overlay
assays using GST-
1-syntrophin-(69-201), which contains the PDZ
domain, as a probe. Rat brain extracts were immunoprecipitated with or
without anti-nNOS antibody (Fig. 3).
Western blotting with anti-nNOS antibody confirmed that nNOS was
precipitated with the antibody (Fig. 3, lane 1).
The same samples were separated by SDS-PAGE, transferred to a
nitrocellulose membrane, and overlaid with GST-
1-syntrophin (Fig. 3,
lanes 3 and 4) or GST
(lanes 5 and 6). The probes were
detected with anti-GST antibody and visualized by enhanced
chemiluminescence. The band corresponding to nNOS was detected when the
immunoprecipitated materials were overlaid with
GST-
1-syntrophin-(69-201) (Fig. 3, lane 3),
but not with GST (lane 5). These results indicate that nNOS can directly interact with
1-syntrophin in rat brain.

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Fig. 3.
Analysis of the interaction between nNOS
and 1-syntrophin by overlay assay. Rat
brain extracts were immunoprecipitated with protein G-Sepharose coupled
with or without anti-nNOS antibody. To confirm that the
immunoprecipitation (IP) was successful, the
immunoprecipitates were analyzed by Western blotting with anti-nNOS
antibody (lanes 1 and 2). The same
samples were subjected to SDS-PAGE, transferred to nitrocellulose
membranes, and overlaid with GST- 1-syntrophin-(69-201) fusion
protein (lanes 3 and 4) or GST
(lanes 5 and 6). The membranes were
then incubated with anti-GST antibody followed by horseradish
peroxidase-labeled anti-rabbit IgG and developed with chemiluminescence
reagent.
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Assessment of the Affinity of nNOS for
1-Syntrophin--
We
further examined the affinity of nNOS for
1-syntrophin in the
presence of different kinds of detergents (Fig.
4). Nonidet P-40 (1%) was used as the
detergent in other analyses, but 1% Nonidet P-40, 0.5% deoxycholate,
and 0.1% SDS (buffer b) or 1% Triton X-100 (buffer
c) also gave similar results as detected by silver staining (Fig.
4A) and Western blotting (Fig. 4B). Deoxycholate (1%) (buffer d) was the strongest condition in which
interaction of nNOS with
1-syntrophin was partly perturbed, whereas
the interaction was not perturbed when deoxycholate was used in
combination with 1% Nonidet P-40 and 0.1% SDS (buffer
b).

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Fig. 4.
Analysis of nNOS-interacting proteins using
various kinds of buffers. A, purification of
nNOS-interacting proteins by GST-nNOS affinity column chromatography
using various kinds of buffers. Adult rat brains were extracted with
the following: buffer a, 1% Nonidet P-40, 500 mM NaCl, 50 mM Tris, pH 7.4, and 1 mM EDTA (lanes 1 and 2);
buffer b, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS,
500 mM NaCl, 50 mM Tris, pH 7.4, and 1 mM EDTA (lanes 3 and 4);
buffer c, 1% Triton, 500 mM NaCl, 50 mM Tris, pH 7.4, and 1 mM EDTA
(lanes 5 and 6); and buffer
d, 0.5% deoxycholate, 100 mM NaCl, 50 mM
Tris, pH 7.4, and 1 mM EDTA (lanes 7 and 8). Each extract was loaded onto a glutathione-Sepharose
column previously coupled with GST (lanes 1,
3, 5, and 7) or GST-nNOS-(1-230)
(lanes 2, 4, 6, and
8) and analyzed as described in the legend to Fig.
1A. The arrow denotes 1-syntrophin.
B, affinities of nNOS for 1-syntrophin and for PSD-95.
Rat brain lysates with various kind of buffers as described above were
precipitated with GST or GST-nNOS and then analyzed by Western blotting
with PSD-95 (upper panel) or 1-syntrophin (lower
panel). Arrows denote PSD-95 (upper panel)
and 1-syntrophin (lower panel).
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Because nNOS has been known to interact with PSD-95, we compared the
affinity of nNOS for
1-syntrophin with that for PSD-95 (Fig.
4B). When detergent lysates from rat brain were precipitated with GST-nNOS-conjugated Sepharose,
1-syntrophin was highly
concentrated in the precipitated fractions. PSD-95 was also solubilized
with all the buffers tested and detected in the lysates by Western blotting. But, in contrast to
1-syntrophin, only a small amount of
PSD-95 was precipitated with GST-nNOS. These results suggest that the
affinity of nNOS for
1-syntrophin is higher than that for
PSD-95.
Colocalization of nNOS with
1-Syntrophin in Primary Cultures of
Neuronal Cells--
We next examined whether nNOS and
1-syntrophin
colocalized in neuronal cells by immunocytochemistry. Primary cultures
of neuronal cells were prepared from fetal rat brain and
maintained for 7-10 days in serum-free medium. Most neurons were
double-labeled with anti-nNOS and anti-
1-syntrophin antibodies with
similar subcellular distribution.
1-Syntrophin-like
immunoreactive substances were present in both neuronal cell bodies and
neurites (Fig. 5, A and
C). nNOS-like immunoreactivity was also detected in both neuronal cell bodies and neurites (Fig. 5, B and
E). All these immunoreactivities became very weak when the
primary antibodies were preincubated with the respective antigens (data
not shown).

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Fig. 5.
Immunofluorescent staining for
1-syntrophin, nNOS, and synaptotagmin in primary
cultures of fetal rat brain neuronal cells. Primary cultures of
neuronal cells were prepared from embryonic day 18 rat brain and
cultured for 10 days. Cells were then fixed with paraformaldehyde and
subjected to immunofluorescent staining as follows. Double-labeling
experiments with anti- 1-syntrophin polyclonal antibody
(A) and anti-nNOS monoclonal antibody (B), those
with anti- 1-syntrophin polyclonal antibody (C) and
anti-synaptotagmin monoclonal antibody (D), and those with
anti-nNOS polyclonal antibody (E) and anti-synaptotagmin
monoclonal antibody (F) were carried out.
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We further examined the distribution of synaptotagmin, an essential
component of the synaptic membranes, to determine the location of
presynaptic structures in these cells. Synaptotagmin-like immunoreactivity was found as punctate signals along the neurites (Fig.
5, D and F) and was not detected in their cell
bodies in most neurons. Double staining with anti-synaptotagmin and
anti-
1-syntrophin antibodies confirmed that syntrophin was not
restricted to synapses (Fig. 5, C and D). Double
staining with anti-synaptotagmin and anti-nNOS antibodies showed that a
fraction of nNOS was colocalized in synapses, but the majority of nNOS
seemed to be present outside of synapses (Fig. 5, E and
F).
Distribution of nNOS and
1-Syntrophin in the
Hypothalamus--
Relative amounts of nNOS and
1-syntrophin
localized in various brain regions were examined by Western blotting
with the respective antibodies. nNOS was highly expressed in the
cerebellum and olfactory bulb as reported previously (5), but low level
expression was detected in all other regions tested, including the
striatum, cerebral cortex, SCN, and hypothalamic paraventricular
nucleus (PVN) (Fig. 6).
1-Syntrophin
was also present in all regions tested, with the highest expression
observed in the PVN (Fig. 6).

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Fig. 6.
Distribution of
1-syntrophin and nNOS in rat brain. Rat brains
were cut into 0.5-mm slices, and tissues were excised from various
regions in the slices as indicated. Extracts from the tissues (10 µg/lane) were analyzed by Western blotting with anti-nNOS
(upper panel) or anti- 1-syntrophin (lower
panel) antibody. The data are representative of five independent
experiments.
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Immunohistochemical staining of rat hypothalamic sections using
anti-nNOS and anti-
1-syntrophin antibodies was done. In the hypothalamus,
1-syntrophin-like immunoreactive substance was observed in the SCN, PVN, and anterior hypothalamic area (Fig. 7, A and B).
nNOS-positive neurons were also detected in the SCN and PVN (Fig. 7,
D and E), consistent with previous studies (5, 6). In the SCN, nNOS- and
1-syntrophin-like immunoreactive substances were most concentrated in the dorsomedial region, whereas weak signals were also detected in the ventrolateral region (Fig. 7,
A, B, D, and E). In the PVN,
1-syntrophin- and nNOS-like immunoreactive substances were detected
mainly in the magnocellular part. No immunostaining was detected when
primary antibodies were preincubated with their respective antigens,
confirming the specificity of immunolabeling (Fig. 7, C and
F).

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Fig. 7.
Photomicrographs of brain sections stained
with anti- 1-syntrophin and anti-nNOS
antibodies. Rat brain sections including the hypothalamus were
immunostained with antibodies against 1-syntrophin (A and
B) and nNOS (D and E). Control
experiments using the antibodies preincubated with GST- 1-syntrophin
(C) or GST-nNOS (F), respectively, were done.
OC, optic chiasm; 3V, third ventricle.
Scale bars indicate 200 µm.
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Fine distributions of nNOS and
1-syntrophin in the dorsomedial
region of the SCN were observed using a confocal microscope. In the
SCN, nNOS-like immunoreactive substance was detected in the cell matrix
of neuronal cell bodies, but not in the nuclei (Fig.
8A).
1-Syntrophin also
showed a subcellular distribution similar to nNOS (Fig. 8B).
Superimposing fluorescence images for nNOS and
1-syntrophin gave a
yellow color, suggesting that nNOS and
1-syntrophin are
colocalized in the same regions of the same SCN neurons (Fig.
8C). In the PVN, the colocalization of nNOS with
1-syntrophin was also observed (data not shown).

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Fig. 8.
Colocalization of nNOS and
1-syntrophin in the SCN. Rat brain sections
were double-labeled with antibodies against nNOS (A) and
1-syntrophin (B) and observed with a confocal microscope.
The two fluorescence images were superimposed (C) to see
whether nNOS and 1-syntrophin were colocalized. D
illustrates the region shown in A-C. OC, optic
chiasm; 3V, third ventricle. Scale bars indicate
50 µm.
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DISCUSSION |
In this study, we have purified an nNOS-binding protein from
bovine brain and identified it as
1-syntrophin. We further
demonstrated that nNOS- and
1-syntrophin-like immunoreactive
substances showed similar subcellular distribution in primary cultures
from fetal rat brain. Finally, the two proteins were expressed at
relatively high levels and colocalized in the PVN and SCN in
hypothalamic sections of adult rat brains. These results suggest that
nNOS interacts with
1-syntrophin in specific neurons in brain.
nNOS was originally found in mammalian brain, but was later shown to be
present also in skeletal muscle, lung epithelial cells, and certain
endocrine glands (12, 19). In skeletal muscle, nNOS is localized at the
sarcolemmal membranes by association with syntrophins, a component of
the dystrophin complex (11, 20). The dystrophin complex is a membrane
cytoskeletal structure that links the sarcolemmal membranes to
extracellular matrix proteins and intracellular actin fibers. In brain,
on the other hand, nNOS has been shown to be associated with PSD-95 and
PSD-93 (11), which are localized at the synapses as a component of the
postsynaptic density structures (10). But the distribution of nNOS is
found not only in the synapses, but also in the entire surfaces of cell bodies and neurites in several types of neurons such as those in the
PVN of the hypothalamus (5). These findings indicate that there are
different mechanisms that define the intracellular localization of nNOS
depending on cell types.
Our present data showed that
1-syntrophin was associated with nNOS
in vitro even in the presence of strong detergents such as
Nonidet P-40, deoxycholate, and SDS. The binding affinity of nNOS for
1-syntrophin seemed to be much higher than that for PSD-95 as
estimated by pull-down assay. In addition, nNOS and
1-syntrophin
were colocalized in cell bodies, neuronal processes, and synapses in
cultured neurons from fetal rat brains. These two proteins were also
colocalized in neuronal cells in the SCN and PVN. From these results,
we propose that
1-syntrophin also contributes to determining the
subcellular localization of nNOS in certain regions of the brain.
1-Syntrophin was expressed in most regions in adult rat brain
judging from Western blotting with anti-
1-syntrophin antibody. The
present immunohistochemical data showed that the level of
1-syntrophin was relatively high in several neurons, including the
PVN and SCN in the hypothalamus (Fig. 7). These two nuclei have been
known to contain nNOS. These results support the possibility that nNOS
and
1-syntrophin interact with each other in certain brain regions
and imply that they have functional relationships. The
immunohistochemical distribution of
1-syntrophin is not completely consistent with its mRNA distribution previously shown by in
situ hybridization (21), but the discrepancy might be elicited by the stability of mRNA and protein.
In skeletal muscle, syntrophins have been shown to be a component of
the dystrophin complex and to function as molecular adaptors that
recruit signaling proteins to the membrane (14). Dystrophin is also
present in brain and has been reported to localize at postsynaptic
densities (22). However, nNOS is unlikely to interact with dystrophin
in brain because nNOS is membrane-associated even in the brains of
mdx mice that lack dystrophin (20). Rather, nNOS might be
associated with membrane proteins such as other dystrophin family
proteins via
1-syntrophin.
The SCN and PVN have key roles in the regulation of metabolism and
behavior through controlling the autonomic nervous system and endocrine
functions in mammals. The PVN regulates various neuroendocrine hormones
through the hypothalamohypophysial system, and NO has been suggested to
be implicated in its function (23). The SCN is the nucleus containing
the circadian oscillator responsible for circadian rhythms (24) and a
mechanism controlling the autonomic nervous system (7). We have
previously shown that infusion of
NG-methylarginine into the third ventricle
in rats disrupts the circadian rhythm of drinking behavior (8),
suggesting that NO might be involved in the generation and/or
synchronization of the circadian oscillator. nNOS- and
1-syntrophin-like immunoreactive substances were detected at
relatively high levels in the dorsomedial region of the SCN, where
vasopressin-containing neurons exist, and it was shown that these
vasopressin neurons are involved in the regulation of secretion of
adrenal glucocorticoid (25). Therefore, it will be interesting to
examine the coexistence of nNOS and
1-syntrophin with vasopressin in
the SCN to obtain further information on the physiological functions of
nNOS and
1-syntrophin in the SCN.