Department of Pharmacology, Johannes Gutenberg University, D-55101 Mainz, Germany
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ABSTRACT |
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In mammalian skeletal muscle,
neuronal-type nitric oxide synthase (nNOS) is found to be enriched at
neuromuscular endplates. Here we demonstrate the colocalization of the
nicotinic acetylcholine receptor (nAChR, stained with -bungarotoxin)
and nNOS (stained with a specific antibody) in murine
C2C12 myotubes. However, coimmunoprecipitation experiments demonstrated no evidence for a direct protein-protein association between the nAChR and nNOS in C2C12
myotubes. An antibody to the
1-subunit of the nAChR did
not coprecipitate nNOS, and an nNOS-specific antibody did not
precipitate the
1-subunit of the nAChR. Treatment of
mice with bacterial LPS downregulated the expression of nNOS in
skeletal muscle, and treatment of C2C12 cells
with bacterial LPS and interferon-
markedly decreased nNOS mRNA and
protein expression. In contrast, mRNA and protein of the nAChR (
-,
-, and
-subunits) remained unchanged at the mRNA and protein
levels. These data demonstrate that nNOS and the nAChR are colocalized
in murine skeletal muscle and C2C12 cells but differ in their expressional regulation.
nitric oxide synthase I; nicotinic acetylcholine receptor; colocalization; expressional regulation; ribonuclease protection analysis
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INTRODUCTION |
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NITRIC OXIDE (NO) seems to have a functional role both in the development of skeletal muscle and in muscle function. At developing neuromuscular junctions (NMJ), myocyte-derived NO functions as a retrograde messenger for feedback inhibition of polyneuronal innervation (42). During muscle maturation, the fusion process of myoblasts to form multinucleated myotubes has been found to be NO dependent (22). In mature skeletal muscle, NO has been shown to reduce contractile force (13, 20) and to modulate uptake and metabolism of glucose (9, 34, 45). Most of these NO effects have been attributed to constitutive, Ca2+-dependent NO synthase (NOS) activity. In 1993, Nakane et al. (28) reported that mRNA and protein of the neuronal-type NOS (nNOS) are expressed at high levels in skeletal muscle. Further studies showed that in skeletal muscle, nNOS is almost exclusively associated with the sarcolemma and concentrated at neuromuscular endplates (3, 13, 21, 31). The submembranous localization of nNOS in skeletal muscle has been attributed to an NH2-terminal PDZ protein interaction motif, linking nNOS to the dystrophin-glycoprotein complex (DGC) of the extrajunctional sarcolemma (2, 3, 18). Additionally, nNOS has been shown to interact directly with muscle-specific caveolin-3 (41), which is also a member of the DGC (26, 40). However, the molecular basis for the accumulation of nNOS protein at neuromuscular endplates is still not completely clear. Therefore, one aim of this study was to investigate whether nNOS and the nicotinic acetylcholine receptor (nAChR) are linked by a direct protein-protein interaction.
There are disease states that are associated with a significant downregulation of nNOS. For example, in autoimmune myasthenia gravis, nNOS and utrophin are markedly reduced at the NMJ (19, 39, 44). In addition, the disease proceeds with a marked loss of membrane nAChRs (11). Whether these phenomena are related is not known at this time. Therefore, the current study also investigates whether a downregulation of the expression of nNOS at the NMJ is associated with a similar regulation of the nAChR.
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EXPERIMENTAL PROCEDURES |
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Cell culture.
C2C12 myoblasts are a subclone of the myogenic
C2 cell line derived from regenerating skeletal muscle of
adult C3H mice (43) and can be differentiated into
spontaneously contracting myotubes under low-mitogen conditions
(38). C2C12 myoblasts were
obtained from the American Type Culture Collection (Manassas, VA) and
grown under subconfluent conditions (~70% confluence) in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, penicillin, and streptomycin at 37°C in a
humidified air atmosphere with 10% CO2. For
differentiation into myotubes, myoblasts (up to passage 10)
were allowed to grow to confluence. The growth medium was then replaced
by low-mitogen medium (DMEM supplemented with 3% horse serum;
Invitrogen). The first fused cells were observed 24-36 h later.
Treatment of cells with LPS (2 µg/ml) and/or IFN- (100 U/ml) was
performed on day 4 of differentiation, when the culture
consisted mainly of multinucleated spontaneously contracting myotubes.
The mRNA of nNOS and subunits of the nAChR was determined 10 h
later, and protein was determined 20 h later. Initial time course
studies had indicated that the changes were most apparent at these
points in time.
Double-fluorescence labeling of cells.
C2C12 cells were cultured on multitest glass
slides (Bibby Dunn, Asbach, Germany) coated with a solution of 0.0025%
calf skin collagen and 0.005% gelatin (both Merck Eurolab, Darmstadt,
Germany) in phosphate-buffered saline (PBS; incubation time 3 h). All subsequent incubations were carried out in the dark. Unfixed cells were
first incubated with Texas Red-conjugated
-bungarotoxin (1 µg/ml
in PBS; Molecular Probes, Leiden, The Netherlands) for 1 h, washed
in PBS, and fixed in an ice-cold mixture of acetone-methanol (50:50,
vol/vol) for 30 s. Slides were then air-dried, washed in PBS, and
incubated in blocking solution (4% dry milk powder in PBS) for 30 min.
Thereafter, slides were washed again in PBS and incubated with a
polyclonal anti-nNOS antibody (Ref. 36; 1:500 in PBS) or
nonimmune rabbit serum overnight at 4°C. Slides were then washed
three times in PBS and incubated with the secondary fluorescein-conjugated anti-rabbit antibody (1:50 in PBS; D. N. Camon Misgav Yaad, Israel) for 1 h at room temperature. Finally, slides were washed in PBS, mounted in glycerol gelatin, and
coverslipped. Fluorescence was analyzed with the Leitz DM RB
fluorescence microscope.
Animals and tissues. Male SPF Balb/c mice were obtained from Charles River (Sulzfeld, Germany). Some animals were treated with an intraperitoneal injection of 7.5 mg/kg LPS dissolved in PBS; control animals received PBS alone. Animals were killed 6 h later by cervical dislocation, and brain and muscle tissues were rapidly removed and frozen in liquid nitrogen.
Cloning of mouse-specific cDNA fragments and generation of
antisense RNA.
Total RNA was isolated either by guanidinium isothiocyanate
(GIT)-phenol-chloroform extraction (for cultured cells; Ref.
5) or by centrifugation through cesium chloride (for
tissues; Ref. 35). Total RNA was isolated from murine
cerebellum (for nNOS), murine RAW 264.7 macrophages (for -actin),
C2C12 myotubes (for nAChR
1- and
-subunits), and murine gastrocnemius muscle (for nAChR
-subunit).
Three micrograms of these RNAs were annealed with one-half microgram of
oligo(dT) primer (Amersham Biosciences, Freiburg, Germany) and
reverse-transcribed at room temperature with SuperScript reverse
transcriptase (Invitrogen) in a total volume of twenty microliters
according to the instructions of the manufacturer. The reaction was
performed at 42°C for 50 min and stopped by heating at 72°C for 15 min. The resulting cDNA (3 µl of the reverse transcription product)
was amplified by polymerase chain reaction (PCR) in 50 µl of
Taq polymerase buffer consisting of 60 mM
Tris · HCl pH 10.0, 15 mM ammonium sulfate, 1.5 mM MgCl2, 0.2 mM dNTPs, 50 pmol oligonucleotide primers,
and 2.5 U of Taq polymerase (Amersham Biosciences).
Amplification of cDNA for nNOS and
-actin was performed with 35 cycles (1 min at 95°C, 1 min at 60°C, and 2 min at 72°C) after
the initial denaturation step (5 min at 95°C). The final extension
period at 72°C was 10 min. The following oligonucleotides were used:
for nNOS (cf. the murine nNOS cDNA; Ref. 29)
5'-ACCATCTTCCAGGCCTTCAAGTAC-3' (bp 3363-3386) as the sense primer
and 5'-TGGACTCAGATCTAAGGCGGTTG-3' (bp 4335-4313) as the antisense
primer; for
-actin (cf. the bovine actin cDNA, GenBank accession no.
K00622; Ref. 7) 5'-ACCAACTGGGACGACATGGAG-3' (bp 1-21)
as the sense primer and 5'-CGTGAGGATCTTCATGAGGTAGTC-3' (bp
354-331) as the antisense primer. Amplification products were 973 bp for nNOS and 354 bp for
-actin.
RNase protection analyses with mouse-specific cRNA probes.
RNase protection analyses were performed as described previously
(35). Briefly, radiolabeled cRNA probes were generated by
in vitro transcription of 0.5 µg of plasmid DNA with T3 or T7 RNA
polymerase (Amersham Biosciences) and 90 µCi of
[-32P]UTP per reaction (incubation for 1 h).
Thereafter, template DNA was degraded by incubation with DNase I for 15 min and labeled cRNA probes were purified on NucTrap probe purification
columns (Stratagene). For each hybridization, 40 µg of total RNA from C2C12 cells or 20 µg of total RNA from
tissues were incubated overnight at 51°C with 200,000 cpm of nNOS
cRNA probe, 200,000 cpm of nAChR
1-,
-, or
-subunit cRNA probes, and 30,000 cpm of
-actin cRNA probe.
Unhybridized probe was then digested by treatment with a mixture of
RNase A and RNase T1 (Roche Diagnostics, Mannheim, Germany). RNase
activity was stopped by the addition of proteinase K in a buffer
containing 10% sodium dodecyl sulfate (SDS). Protected fragments were
extracted with phenol-chloroform, precipitated with ethanol, and
separated electrophoretically in a denaturing 6% polyacrylamide gel
containing 8 M urea. DNA fragments derived from pGl2-Basic restricted
with HinfI (for detection of nNOS mRNA) or from pUC19
restricted with Sau3A (for detection of nAChR subunits) were
labeled with [
-32P]ATP and served as size markers.
Densitometric analyses of gels were performed with a Molecular Imager
(Bio-Rad, Munich, Germany), and results were quantified by comparison
with the hybridization signal of a
-actin cRNA probe.
Protein fractionation and Western blotting.
Cells or tissues were washed with PBS. Two milliliters of ice-cold RIPA
buffer were then added [50 mM Tris · HCl, pH 8, 150 mM NaCl, 1% (vol/vol) NP-40, 0.1% (vol/vol) SDS, 10 µg/ml
pepstatin, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.2 mM
phenylmethylsulfonyl fluoride] and incubated for 30 min on ice while
shaking. The cell fragments were scraped off the dish and centrifuged
at 10,000 g for 10 min at 4°C. Tissues were homogenized
with an Ultra Turrax homogenizer. Proteins were separated by denaturing
polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto
nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) in a
semidry electrophoretic transfer cell (Trans-Blot, Bio-Rad). Blots were blocked for 60 min with 5% dry milk powder and 0.05% Tween 20 in
Tris-buffered saline (TBS). Then, after three washes (5 min each) in
TBS containing 1% bovine serum albumin and 0.1% Tween 20, blots were
incubated overnight at 4°C with the primary antibodies: a rabbit
polyclonal antibody (36) or a mouse monoclonal antibody to
nNOS (1:250), a mouse monoclonal antibody to the nAChR
1-subunit (1:250), or a mouse monoclonal to utrophin
(1:250) (all BD Biosciences, Heidelberg, Germany). Thereafter, blots
were washed in a solution of 5% dry milk powder in TBS, containing
0.05% Tween 20 (3 times for 5 min) and incubated with the secondary
antibody conjugated to alkaline phosphatase (Sigma, Deisenhofen,
Germany; 60 min at room temperature). After a final wash step in
TBS-0.05% Tween 20, immunoreactive proteins were visualized with nitro
blue tetrazolium (NBT)-X-phosphate.
Immunoprecipitation studies.
For immunoprecipitation, 1-ml aliquots of RIPA buffer-solubilized
homogenates were precleared by incubation with 50 µl of normal rabbit
serum (1 h on ice on a rotating shaker). Twenty microliters of Protein
G Plus-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA) were then
added, and the incubation continued for another 30 min. Supernatants of
the subsequent centrifugation (10,000 g, 15 min, 4°C) were
incubated with 2.5 µg/ml of the antigen-specific antibody overnight
at 4°C (rotating shaker). A rabbit polyclonal antibody to nNOS
(36), a mouse monoclonal antibody to the nAChR 1-subunit, or a mouse monoclonal antibody to utrophin
(both BD Biosciences) were used. For control purposes, precipitating
antibodies were replaced with mouse or rabbit IgGs (5 µg/ml, Santa
Cruz Biotechnology). After complexed material was removed by
centrifugation (10,000 g, 10 min), the resulting
supernatants were incubated for 90 min (rotating shaker) with 20 µl
of Protein A- or Protein G Plus-Agarose, respectively.
Immunoprecipitates were collected by centrifugation at 10,000 g for 5 min and washed twice with RIPA buffer. After the
final wash with TBS, immunoprecipitates were pelleted by centrifugation at 10,000 g for 5 min. Pellets were resuspended in 20 µl
of Laemmli buffer, boiled for 5 min, and centrifuged (10,000 g for 5 min) to pellet the agarose beads. Supernatants
(total immunoprecipitated proteins) were analyzed with SDS-PAGE and
subsequent Western blotting. For detection of nNOS, nAChR
1-subunit or utrophin blots were incubated with the
respective monoclonal primary antibodies described for the Western
blotting procedure.
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RESULTS |
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Colocalization of nNOS with the 1-subunit of the
nAChR in C2C12 myotubes.
In double-fluorescence studies on C2C12
myotubes, nNOS was found concentrated in areas that also showed a
strong labeling for
-bungarotoxin, identifying them as accumulations
of nAChRs (Fig. 1).
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No evidence for a direct protein-protein interaction between nNOS
and the nAChR 1-subunit in C2C12
myotubes.
In immunoprecipitation experiments with a precipitating antibody
specific to the nAChR
1-subunit, the
-subunit of the
nAChR was detected by Western blots in all precipitates (Fig.
2A). However, when an
anti-nNOS antibody was used for the immunoprecipitation, no band
corresponding to the nAChR
1-subunit could be detected (Fig. 2A). Conversely, with an anti-nNOS antibody, nNOS
protein could be precipitated and detected in subsequent Western blots, whereas no band was found when the blots were probed with an antibody to the nAChR
1-subunit (n = 5; not
shown). When an anti-utrophin antibody was used for precipitation,
neither nNOS nor the nAChR
1-subunit could be detected
in subsequent Western blots (n = 4; not shown).
However, the anti-utrophin antibody detected a single band of 400 kDa
in Western blots with C2C12 cell protein (n = 2; not shown). When the precipitating antibodies
were replaced with mouse or rabbit IgG, no specific band could be
detected in subsequent Western blots with either an anti-nNOS antibody
or an anti-nAChR
1 antibody (Fig. 2B).
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Expression of nNOS mRNA and mRNA for nAChR subunits in murine
skeletal muscle and C2C12 myotubes: effect of
LPS or LPS-IFN-.
nNOS mRNA is significantly expressed in diaphragm and gastrocnemius
muscle of untreated mice (Fig.
3A) and also in untreated C2C12 myotubes (Fig. 3B). The
expression of nNOS mRNA was strongly reduced in skeletal muscle after
treatment of the mice with LPS (7.5 mg/kg; Fig. 3A) and in
C2C12 myotubes treated with LPS (2 µg/ml) and
IFN-
(100 U/ml) (Fig. 3B). Densitometric quantification of this downregulation revealed a decrease of nNOS mRNA to 45 ± 10% in diaphragm (as compared with controls), to 51 ± 9% in gastrocnemius muscle, and to 30 ± 3% in
C2C12 myotubes (means ± SE;
n = 6). In contrast, treatment of mice with LPS or
treatment of C2C12 myotubes with LPS-IFN-
did not change the expression of the
-,
-, or
-subunits of the
nAChR (n = 4-6; Fig.
4). In agreement with the literature
(10), the
-subunit was not found in adult tissue (data
not shown).
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Protein expression of nNOS and nAChR in
C2C12 myotubes: effect of LPS and IFN-.
When C2C12 myotubes were treated with LPS (2 µg/ml) and IFN-
(100 U/ml), nNOS immunoreactivity was markedly
downregulated (Fig. 5). In contrast,
there was no apparent change in the expression level of nAChRs (Fig.
5). Western blots with protein preparation from untreated and LPS- and
IFN-
-treated C2C12 myotubes also demonstrated a marked downregulation of nNOS protein to 39% ± 6% of
control (n = 4; Fig.
6A), whereas no significant
change was seen for the nAChR
1-subunit after
LPS-IFN-
treatment (97% ± 5%; Fig. 6B). Also in
skeletal muscle of LPS (7.5 mg/kg)-treated mice, nNOS protein
expression decreased significantly; in diaphragm it reached 42% ± 5%
of control (mean ± SE of 3 independent Western blots).
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DISCUSSION |
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nNOS has been found to be enriched at the postjunctional site of
the neuromuscular endplate in slow- and fast-twitch fibers of skeletal
muscle from different species (4, 13, 44). In
C2C12 skeletal myotubes, nNOS has been shown to
cocluster with nAChRs after treatment with active agrin constructs
(24). In the current study, we found nNOS concentrated in
the vicinity of nAChR clusters (even in the absence of agrin; Fig. 1).
Sarcolemmal localization of nNOS has been attributed to an association
with dystrophin via PDZ domain-mediated binding to
1-syntrophin (2). However, at the NMJ,
dystrophin is almost absent from the crests of the postjunctional
folds, i.e., the site where nAChRs accumulate (6, 37). In
these specialized regions, dystrophin is replaced by its homolog
utrophin (30). Utrophin can associate with the nAChR and
may be involved in the stabilization of the synaptic cytoskeleton
(1, 17). Unlike dystrophin, utrophin has been found to
interact with
1- and
2-syntrophins, the
latter also being largely restricted to NMJs (32). Hence,
-syntrophins could link nNOS to nAChRs at the NMJ. However, in
coimmunoprecipitation experiments, we found no direct protein-protein
interactions of nNOS with the nAChR
1-subunit in
myotubes and also in murine skeletal muscle (Fig. 2). This failure is
unlikely to be technical in nature, because the protein directly
immunoprecipitated could always be detected in subsequent Western blots
(cf. Fig. 2, lanes 2 and 3). Furthermore, our
laboratory has successfully performed coimmunoprecipitations with
inducible NOS (iNOS) and caveolin-3 (14). We have also
been able to coimmunoprecipitate nNOS and caveolin-3 from mouse
skeletal muscle (unpublished results), thereby confirming the findings
of others (41). However, attempts to coprecipitate the
nAChR
1-subunit with an anti-utrophin antibody were also
unsuccessful. Thus the molecular basis of the regional association of
the nAChR with nNOS (shown in Fig. 1) remains unclear at this time.
Functionally, the colocalization of nNOS and the nAChR may favor potential effects of NO on acetylcholine release. Lindgren and Laird (23) showed that NO inhibits the evoked release of neurotransmitter at the frog neuromuscular junction. On the other hand, inhibitors of NOS activity increased acetylcholine release at the motor nerve terminals (33). Thus NO produced locally by nNOS may be involved in the physiological modulation of ACh release. Local NO may also modulate the sensitivity of the nAChR or interfere with subsequent signal transduction cascades. Conversely, activation of nAChR has been shown to activate a Ca2+-dependent protein kinase C pathway that, in turn, upregulates nNOS mRNA expression (27). Also, such signaling could be promoted by a close proximity of the nAChR and nNOS.
We reported previously (15) that nNOS is downregulated in
skeletal muscle of guinea pigs treated with LPS. The present study confirms this finding for murine skeletal muscle and shows a similar downregulation of nNOS in murine C2C12 myotubes
after treatment with LPS and IFN- (Figs. 3, 5, and 6).
In certain disease states such as autoimmune myasthenia gravis, the
expression of nNOS as well as utrophin is markedly reduced at the NMJ
(19, 39, 44). In parallel, there is a loss of membrane
nAChRs (11). However, in the current study, the LPS- and
IFN--induced reduction in nNOS expression was not accompanied by a
similar downregulation of the nAChR (Figs. 4-6). This suggests that, although the two proteins are located in close proximity, their
expression is regulated independently. Also, in skeletal muscle from
mdx mice, a dystrophin-deficient animal model for Duchenne
muscular dystrophy, nNOS and syntrophins are strongly reduced or
completely absent from the extrajunctional sarcolemma (2, 3,
32) and the NMJ (4, 16, 44), whereas the nAChRs
have been reported to be normal in number and localization at the
NMJ of muscles from mdx mice (25). This
situation is similar to that observed in the current study, where the
expression of nNOS was downregulated with no change in the expression
of the nAChR.
On the basis of the expression data generated here, one would expect an
unimpaired nAChR function but an impaired nNOS function after
LPS-INF- treatment. However, iNOS is markedly induced under these
conditions and
although occurring in different subcellular compartments
the iNOS-derived NO production is likely to mask the
reduced nNOS-derived NO production. Findings from our and other
laboratories indicate that contractile function of striated muscle is
reduced by NO irrespective of its source (12, 13, 20).
In conclusion, we have shown a close association of nNOS and the nAChR in C2C12 myotubes. However, the molecular basis of this association remains unclear. The colocalization may favor functional effects of NO on acetylcholine release or on nAChR signaling. In certain disease states, the two proteins may be downregulated in parallel, but the results of the current study clearly demonstrate that the expression of the two proteins is not coupled in LPS-induced (sepsislike) disorders.
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ACKNOWLEDGEMENTS |
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We thank Dr. Petra M. Schwarz for carefully reading the manuscript.
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FOOTNOTES |
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This study was supported by the Collaborative Research Center SFB 553 (Project A6 to U. Förstermann) from the Deutsche Forschungsgemeinschaft, Bonn, Germany.
Address for reprint requests and other correspondence: U. Förstermann, Dept. of Pharmacology, Johannes Gutenberg Univ., Obere Zahlbacher Strasse 67, 55101 Mainz, Germany (E-mail: Ulrich.Forstermann{at}Uni-Mainz.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 21, 2002;10.1152/ajpcell.04767.2002
Received 10 October 2002; accepted in final form 10 December 2002.
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