Colocalization but differential regulation of neuronal NO synthase and nicotinic acetylcholine receptor in C2C12 myotubes

Jutta G. Ebert, Marek Zelenka, Ingolf Gath, Ute Gödtel-Armbrust, and Ulrich Förstermann

Department of Pharmacology, Johannes Gutenberg University, D-55101 Mainz, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha 1-subunit of the nAChR did not coprecipitate nNOS, and an nNOS-specific antibody did not precipitate the alpha 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-gamma markedly decreased nNOS mRNA and protein expression. In contrast, mRNA and protein of the nAChR (alpha -, gamma -, and epsilon -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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-gamma (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 alpha -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 beta -actin), C2C12 myotubes (for nAChR alpha 1- and gamma -subunits), and murine gastrocnemius muscle (for nAChR epsilon -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 beta -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 beta -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 beta -actin.

For nAChR subunits, cDNA was amplified with 30 cycles (45 s at 95°C, 1 min at 54°C, and 1.5 min at 72°C with an extension of 2 s/cycle). The primers used for cDNA amplification of murine nAChR subunits were reported previously (8). Sizes of the amplification products were: nAChR alpha 1-subunit, 721 bp; nAChR gamma -subunit, 733 bp; and nAChR epsilon -subunit, 548 bp.

The amplification products were inserted into the EcoRV site of pCR-Script (Stratagene, La Jolla, CA) with the SureClone ligation kit (Amersham Biosciences). The resulting cDNA clones were sequenced by the dideoxy termination method with the T7 sequencing kit (Amersham Biosciences); plasmids were termed pmnNOS, pmactin, pmAChRa, pmAChRe, and pmAChRg. To generate antisense RNA (cRNA) probes for RNase protection analyses, plasmids were restricted with NcoI (pmnNOS), Asp718 (pmactin), HindIII (pmAChRa, pmAChRe), or PvuII (pmAChRg) for in vitro transcription with T3 RNA polymerase (Amersham Biosciences). The linearized plasmids were then extracted with phenol-chloroform, ethanol-precipitated and resuspendend in diethyl pyrocarbonate (DEPC)-treated H2O.

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 [alpha -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 alpha 1-, gamma -, or epsilon -subunit cRNA probes, and 30,000 cpm of beta -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 [gamma -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 beta -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 alpha 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 alpha 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 alpha 1-subunit or utrophin blots were incubated with the respective monoclonal primary antibodies described for the Western blotting procedure.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Colocalization of nNOS with the alpha 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 alpha -bungarotoxin, identifying them as accumulations of nAChRs (Fig. 1).


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Fig. 1.   Double-fluorescence labeling of two individual C2C12 myotubes (left and right) with a selective antibody to neuronal-type nitric oxide synthase (anti-nNOS, A; fluorescein) and alpha -bungarotoxin (which binds to the alpha -subunit of the nicotinic acetylcholine receptor, B; Texas red). After superimposition (C), the yellow signals indicated by arrows demonstrate the colocalization of nNOS immunoreactivity with alpha -bungarotoxin-positive spots (i.e., nicotinic acetylcholine receptors). Magnification is 400-fold in all panels. The photomicrographs of the 2 cells shown are representative of numerous other cells.

No evidence for a direct protein-protein interaction between nNOS and the nAChR alpha 1-subunit in C2C12 myotubes. In immunoprecipitation experiments with a precipitating antibody specific to the nAChR alpha 1-subunit, the alpha -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 alpha 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 alpha 1-subunit (n = 5; not shown). When an anti-utrophin antibody was used for precipitation, neither nNOS nor the nAChR alpha 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 alpha 1 antibody (Fig. 2B).


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Fig. 2.   Immunoprecipitation of nNOS and of the nicotinic acetylcholine receptor (nAChR) alpha -subunit. Homogenates from C2C12 myotubes were immunoprecipitated (IP) either with a monoclonal antibody to nAChR-alpha or a polyclonal antibody to nNOS. Immunoprecipitated proteins were separated on a 10% polyacrylamide gel. The subsequent Western blots were probed with a monoclonal anti-nAChR-alpha antibody (A). Homogenate (100 µg) from C2C12 myotubes was used as the positive control (Co, no IP), yielding the expected band at ~40 kDa. As a negative control, homogenates from C2C12 myotubes were immunoprecipitated with mouse or rabbit IgG and subsequently probed on a Western blot with a monoclonal anti-nAChR-alpha antibody (B). The positive control (Co, no IP) is the same as in A. Results are representative of 4 independent experiments with identical results. The nonspecific band at ~51 kDa corresponds to the IgG heavy chain seen in Western blots after IP. MM, molecular mass.

Expression of nNOS mRNA and mRNA for nAChR subunits in murine skeletal muscle and C2C12 myotubes: effect of LPS or LPS-IFN-gamma . 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-gamma (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-gamma did not change the expression of the alpha -, gamma -, or epsilon -subunits of the nAChR (n = 4-6; Fig. 4). In agreement with the literature (10), the gamma -subunit was not found in adult tissue (data not shown).


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Fig. 3.   RNase protection analyses of nNOS mRNA from murine skeletal muscle [diaphragm (Diaphr) and gastrocnemius muscle (Gastr), A] and from C2C12 myotubes (B). Total RNA was prepared from skeletal muscle of untreated mice (Co) and LPS-treated mice (LPS). RNA was also prepared from untreated (Co) and LPS-IFN-gamma -treated C2C12 myotubes (LPS/IFN-gamma ) and from murine cerebellum (Cereb, used as a positive control for nNOS mRNA). Hybridizations with an antisense-RNA probe for murine beta -actin were used for standardization. Protected RNA fragments (180 nt for nNOS and 108 nt for beta -actin) were separated on 6% denaturing polyacrylamide gels. Molecular size markers (m) and t-RNA-negative controls (t, 20 µg RNA/lane) are also shown. Results are representative of 3 independent experiments yielding the same results.



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Fig. 4.   RNase protection analyses with antisense-RNA probes specific for different subunits of the murine nAChR. Total RNA was prepared from untreated (Co) and LPS-IFN-gamma -treated C2C12 myotubes (LPS/IFN-gamma ; top). RNA was also obtained from murine cerebellum (Cereb, used as a negative control) and gastrocnemius muscle (Gastr) of untreated (Co) and LPS-treated mice (LPS). RNAs were hybridized with probes to the nAChR alpha 1-subunit (nAChR-alpha ; A), the gamma -subunit (nAChR-gamma ; B), or the epsilon -subunit (nAChR-epsilon ; C). Hybridizations with an antisense RNA probe for murine beta -actin were used for standardization. Protected RNA fragments (265 nt for nAChR-alpha , 211 nt for nAChR-gamma , 231 nt for nAChR-epsilon , and 108 nt for beta -actin) were separated on 6% denaturing polyacrylamide gels. t-RNA-negative controls (t; 20 µg RNA/lane) are also shown. The gels are representative of 3 independent experiments yielding the same results.

Protein expression of nNOS and nAChR in C2C12 myotubes: effect of LPS and IFN-gamma . When C2C12 myotubes were treated with LPS (2 µg/ml) and IFN-gamma (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-gamma -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 alpha 1-subunit after LPS-IFN-gamma 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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Fig. 5.   Effect of 20-h treatment of C2C12 myotubes with LPS (2 µg/ml) and IFN-gamma (100 units/ml) on the expression of nNOS and the nAChR. Fluorescence labeling of the myotubes was done with a specific anti-nNOS antibody and with alpha -bungarotoxin for the labeling of the nAChR. Magnification is 400-fold in all panels. After treatment with LPS and IFN-gamma , nNOS immunoreactivity was markedly reduced (bottom left) compared with control cells (top left; cf. Fig. 1). In contrast, there was no obvious change in the alpha -bungarotoxin fluorescence, suggesting that the nAChR was not downregulated by LPS-IFN-gamma . The photomicrographs of the 2 cells shown are representative for numerous other cells.



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Fig. 6.   Western blots of homogenates from untreated and LPS-IFN-gamma -treated C2C12 myotubes. Proteins were separated on 7.5% polyacrylamide gels (for detection of nNOS; A) or 10% polyacrylamide gels (for detection of nAChR alpha 1-subunit; B). Blots were immunostained with a polyclonal antibody to nNOS (A) or a monoclonal antibody to nAChR-alpha (B). C2C12 protein samples were applied at 100 µg/lane (A) or 70 µg/lane (B). An ADP Sepharose-purified cytosolic fraction from murine cerebellum (Cereb, 5 µg/lane) served as a positive control for nNOS; m, molecular mass marker. Results are representative of 4 independent experiments yielding the same results.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 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 beta 1- and beta 2-syntrophins, the latter also being largely restricted to NMJs (32). Hence, beta -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 alpha 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 alpha 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-gamma (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-gamma -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-gamma 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.


    ACKNOWLEDGEMENTS

We thank Dr. Petra M. Schwarz for carefully reading the manuscript.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Blake, DJ, Tinsley JM, and Davies KE. Utrophin---a structural and functional comparison to dystrophin. Brain Pathol 6: 37-47, 1996[ISI][Medline].

2.   Brenman, JE, Chao DS, Gee SH, Mcgee AW, Craven SE, Santillano DR, Wu ZQ, Huang F, Xia HH, Peters MF, Froehner SC, and Bredt DS. Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha 1-syntrophin mediated by PDZ domains. Cell 84: 757-767, 1996[ISI][Medline].

3.   Brenman, JE, Chao DS, Xia HH, Aldape K, and Bredt DS. Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy. Cell 82: 743-752, 1995[ISI][Medline].

4.   Chao, DS, Silvagno F, Xia H, Cornwell TL, Lincoln TM, and Bredt DS. Nitric oxide synthase and cyclic GMP-dependent protein kinase concentrated at the neuromuscular endplate. Neuroscience 76: 665-672, 1997[ISI][Medline].

5.   Chomczynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

6.   Colledge, M, and Froehner SC. Signals mediating ion channel clustering at the neuromuscular junction. Curr Opin Neurobiol 8: 357-363, 1998[ISI][Medline].

7.   Degen, JL, Neubauer MG, Degen SJ, Seyfried CE, and Morris DR. Regulation of protein synthesis in mitogen-activated bovine lymphocytes. Analysis of actin-specific and total mRNA accumulation and utilization. J Biol Chem 258: 12153-12162, 1983[Abstract/Free Full Text].

8.   Drescher, DG, Khan KM, Green GE, Morley BJ, Beisel KW, Kaul H, Gordon D, Gupta AK, Drescher MJ, and Barretto RL. Analysis of nicotinic acetylcholine receptor subunits in the cochlea of the mouse. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 112: C267-C273, 1995.

9.   Etgen, GJ, Fryburg DA, and Gibbs EM. Nitric oxide stimulates skeletal muscle glucose transport through a calcium/contraction- and phosphatidylinositol-3-kinase-independent pathway. Diabetes 46: 1915-1919, 1997[Abstract].

10.   Evans, S, Goldman D, Heinemann S, and Patrick J. Muscle acetylcholine receptor biosynthesis. Regulation by transcript availability. J Biol Chem 262: 4911-4916, 1987[Abstract/Free Full Text].

11.   Fambrough, DM, Drachman DB, and Satyamurti S. Neuromuscular junction in myasthenia gravis: decreased acetylcholine receptors. Science 182: 293-295, 1973[ISI][Medline].

12.   Galler, S, Hilber K, and Gobesberger A. Effects of nitric oxide on force-generating proteins of skeletal muscle. Pflügers Arch 434: 242-245, 1997[ISI][Medline].

13.   Gath, I, Closs EI, Gödtel-Armbrust U, Schmitt S, Nakane M, Wessler I, and Förstermann U. Inducible NO synthase II and neuronal NO synthase I are constitutively expressed in different structures of guinea pig skeletal muscle: implications for muscle function. FASEB J 10: 1614-1620, 1996[Abstract/Free Full Text].

14.   Gath, I, Ebert J, Gödtel-Armbrust U, Reske-Kunz AB, Ross R, and Förstermann U. NO synthase II in mouse skeletal muscle is associated with caveolin 3. Biochem J 340: 723-728, 1999[ISI][Medline].

15.   Gath, I, Gödtel-Armbrust U, and Förstermann U. Expressional downregulation of neuronal-type NO synthase I in guinea pig skeletal muscle in response to bacterial lipopolysaccharide. FEBS Lett 410: 319-323, 1997[ISI][Medline].

16.   Grozdanovic, Z, and Gossrau R. Co-localization of nitric oxide synthase I (NOS I) and NMDA receptor subunit 1 (NMDAR-1) at the neuromuscular junction in rat and mouse skeletal muscle. Cell Tissue Res 291: 57-63, 1998[ISI][Medline].

17.   Guo, WXA, Nichol M, and Merlie JP. Cloning and expression of full length mouse utrophin---the differential association of utrophin and dystrophin with AChR clusters. FEBS Lett 398: 259-264, 1996[ISI][Medline].

18.   Hendriks, W. Nitric oxide synthase contains a discs-large homologous region (DHR) sequence motif. Biochem J 305: 687-688, 1995[ISI][Medline].

19.   Ito, H, Yoshimura T, Satoh A, Takino H, Tsujihata M, and Nagataki S. Immunohistochemical study of utrophin and dystrophin at the motor end-plate in myasthenia gravis. Acta Neuropathol (Berl) 92: 14-18, 1996[Medline].

20.   Kobzik, L, Reid MB, Bredt DS, and Stamler JS. Nitric oxide in skeletal muscle. Nature 372: 546-548, 1994[ISI][Medline].

21.   Kusner, LL, and Kaminski HJ. Nitric oxide synthase is concentrated at the skeletal muscle endplate. Brain Res 730: 238-242, 1996[ISI][Medline].

22.   Lee, KH, Baek MY, Moon KY, Song WK, Chung CH, Ha DB, and Kang MS. Nitric oxide as a messenger molecule for myoblast fusion. J Biol Chem 269: 14371-14374, 1994[Abstract/Free Full Text].

23.   Lindgren, CA, and Laird MV. Nitroprusside inhibits neurotransmitter release at the frog neuromuscular junction. Neuroreport 5: 2205-2208, 1994[ISI][Medline].

24.   Lück, G, Hoch W, Hopf C, and Blottner D. Nitric oxide synthase (NOS-1) coclustered with agrin-induced AChR-specializations on cultured skeletal myotubes. Mol Cell Neurosci 16: 269-281, 2000[ISI][Medline].

25.   Lyons, PR, and Slater CR. Structure and function of the neuromuscular junction in young adult mdx mice. J Neurocytol 20: 969-981, 1991[ISI][Medline].

26.   McNally, EM, Moreira ED, Duggan DJ, Lisanti MP, Lidov HGW, Vainzof M, Bonnemann CG, PassosBueno MR, Hoffman EP, Zatz M, and Kunkel LM. Caveolin-3 in muscular dystrophy. Hum Mol Genet 7: 871-877, 1998[Abstract/Free Full Text].

27.   Nakamura, K, Takahashi T, Taniuchi M, Hsu CX, and Owyang C. Nicotinic receptor mediates nitric oxide synthase expression in the rat gastric myenteric plexus. J Clin Invest 101: 1479-1489, 1998[Abstract/Free Full Text].

28.   Nakane, M, Schmidt HHHW, Pollock JS, Förstermann U, and Murad F. Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle. FEBS Lett 316: 175-180, 1993[ISI][Medline].

29.   Ogura, T, Yokoyama T, Fujisawa H, Kurashima Y, and Esumi H. Structural diversity of neuronal nitric oxide synthase mRNA in the nervous system. Biochem Biophys Res Commun 193: 1014-1022, 1993[ISI][Medline].

30.   Ohlendieck, K, Ervasti JM, Matsumura K, Kahl SD, Leveille CJ, and Campbell KP. Dystrophin-related protein is localized to neuromuscular junctions of adult skeletal muscle. Neuron 7: 499-508, 1991[ISI][Medline].

31.   Oliver, L, Goureau O, Courtois Y, and Vigny M. Accumulation of NO synthase (type-1) at the neuromuscular junctions in adult mice. Neuroreport 7: 924-926, 1996[ISI][Medline].

32.   Peters, MF, Adams ME, and Froehner SC. Differential association of syntrophin pairs with the dystrophin complex. J Cell Biol 138: 81-93, 1997[Abstract/Free Full Text].

33.   Ribera, J, Marsal J, Casanovas A, Hukkanen M, Tarabal O, and Esquerda JE. Nitric oxide synthase in rat neuromuscular junctions and in nerve terminals of Torpedo electric organ-its role as regulator of acetylcholine release. J Neurosci Res 51: 90-102, 1998[ISI][Medline].

34.   Roberts, CK, Barnard RJ, Scheck SH, and Balon TW. Exercise-stimulated glucose transport in skeletal muscle is nitric oxide dependent. Am J Physiol Endocrinol Metab 273: E220-E225, 1997[Abstract/Free Full Text].

35.   Sambrook, J, Fritsch EF, and Maniatis T. Molecular Cloning. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989.

36.   Schmidt, HHHW, Gagne GD, Nakane M, Pollock JS, Miller MF, and Murad F. Mapping of neural nitric oxide synthase in the rat suggests frequent co-localization with NADPH diaphorase but not with soluble guanylyl cyclase, and novel paraneural functions for nitrinergic signal transduction. J Histochem Cytochem 40: 1439-1456, 1992[Abstract/Free Full Text].

37.   Sealock, R, Butler MH, Kramarcy NR, Gao KX, Murnane AA, Douville K, and Froehner SC. Localization of dystrophin relative to acetylcholine receptor domains in electric tissue and adult and cultured skeletal muscle. J Cell Biol 113: 1133-1144, 1991[Abstract].

38.   Silberstein, L, Inestrosa NC, and Hall ZW. Aneural muscle cell cultures make synaptic basal lamina components. Nature 295: 143-145, 1982[ISI][Medline].

39.   Slater, CR, Young C, Wood SJ, Bewick GS, Anderson LV, Baxter P, Fawcett PR, Roberts M, Jacobson L, Kuks J, Vincent A, and Newsom Davis J. Utrophin abundance is reduced at neuromuscular junctions of patients with both inherited and acquired acetylcholine receptor deficiencies. Brain 120: 1513-1531, 1997[Abstract].

40.   Song, KS, Scherer PE, Tang ZL, Okamoto T, Li SW, Chafel M, Chu C, Kohtz DS, and Lisanti MP. Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells---caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins. J Biol Chem 271: 15160-15165, 1996[Abstract/Free Full Text].

41.   Venema, VJ, Ju H, Zou R, and Venema RC. Interaction of neuronal nitric-oxide synthase with caveolin-3 in skeletal muscle. Identification of a novel caveolin scaffolding/inhibitory domain. J Biol Chem 272: 28187-28190, 1997[Abstract/Free Full Text].

42.   Wang, T, Xie ZP, and Lu B. Nitric oxide mediates activity-dependent synaptic suppression at developing neuromuscular synapses. Nature 374: 262-266, 1995[ISI][Medline].

43.   Yaffe, D, and Saxel O. Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270: 725-727, 1977[ISI][Medline].

44.   Yang, CC, Alvarez RB, Engel WK, Haun CK, and Askanas V. Immunolocalization of nitric oxide synthases at the postsynaptic domain of human and rat neuromuscular junctions---light and electron microscopic studies. Exp Neurol 148: 34-44, 1997[ISI][Medline].

45.   Young, ME, Radda GK, and Leighton B. Nitric oxide stimulates glucose transport and metabolism in rat skeletal muscle in vitro. Biochem J 322: 223-228, 1997[ISI][Medline].


Am J Physiol Cell Physiol 284(4):C1065-C1072
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