Nitric oxide synthase is concentrated at the skeletal muscle endplate

BRAIN RESEARCH ELSEVIER

Brain Research 730 (1996) 238-242

Short communication

Nitric oxide synthase is concentrated at the skeletal muscle endplate Linda L. Kusner, Henry J. Kaminski * Department of Neurology, Case Western Reser~,e Unit'ersiO' School of Medicine, Unil ersity Hospitals of Clel,eland, Department ~/ Veterans A~l~lirs Medical Center in Cleceland, 11100 Euclid At'enue, Clet:eland, OH 44106. USA Accepted 28 May 1996

Abstract

NO performs a wide array of cell signaling functions. Neuronal NO synthase (nNOS) immunoreactivity and nicotinamide adenine dinucleotide phosphate diaphorase (NDP) activity, a marker of nNOS, were concentrated at adult rat neuromuscular junctions and persisted in denervated muscle indicating the localization of the enzyme to the postsynaptic surface. The concentration of nNOS at the muscle endplate suggests NO could serve as a messenger pre- and postsynapticly. Keywords: Neuromuscular junction; Synaptic transmission; Nitric oxide; Cholinergic transmission; Skeletal muscle: Ocular muscle: Acetylcholine receptor

The neuromuscular junction consists of highly specialized pre- and postsynaptic structures designed for rapid translation of electrical to chemical signals [5]. It has become evident that the nerve and muscle communicate in an anterograde and retrograde fashion beyond the simple release of the transmitter, acetylcholine. Nitric oxide (NO) is a prime candidate to function as such a signal at the neuromuscular junction. In the nervous system NO is involved in synaptic plasticity and acts as a retrograde signal to enhance transmitter release from hippocampal neurons [1,19]. Autonomic nerves modulate intestinal motility by NO-mediated relaxation of smooth muscle [10,11]. NO donors suppress synaptic currents in nerve and myocyte co-cultures, and this property may allow NO to serve in synapse elimination during synaptogenesis [21]. NO is a free radical gas which is not easily localized in tissue, and therefore, the majority of investigations focus on identification of NO synthase (nNOS), the enzyme which produces NO, as presumptive evidence of NO activity [1,19]. nNOS reduces nitroblue tetrazolium (NBT) in a nicotinamide adenine dinucleotide phosphate (NADPH)dependent fashion which requires calcium and calmodulin. In select neuronal populations, the reduction of NBT had been used to localize NADPH diaphorase (NDP), which has been identified as nNOS [3,15]. nNOS is expressed in high levels in adult skeletal

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muscle [16]. In particular, nNOS is concentrated in the sarcolemma of fast twitch fibers where NO appears to modulate contractile force and promote relaxation [12]. Because of NO's function as a transmitter in the nervous system and the presence of nNOS in skeletal muscle, we hypothesized that nNOS may be concentrated at the mature neuromuscular junction where NO could participate in a host of synaptic functions. To evaluate the distribution of nNOS at the neuromuscular junction, we performed NDP histochemistry and nNOS immunohistochemistry on skeletal and extraocular muscle. Adult Lewis rat extensor digitorum longus (EDL, a muscle composed primarily of fast-twitch fibers), soleus (a predominantly slow-twitch muscle), and extraocular rectus muscles were sandwiched in liver and frozen in liquid N~ cooled isobutane. NDP histochemistry was performed by the method of Dawson et al. [3] with modifications to emphasize staining of endplate regions and minimize sarcolemmal staining. Ten Ixm cryostat sections were dried and fixed in 2% paraformaldehyde in phosphate buffered saline, pH 7.4 (PBS). After a rinse in PBS, sections were incubated in 0.2% Triton X-100 for 10 min at 37°C. The reaction was performed for 1 h in a dark, humidified chamber at 37°C in 0.2% Triton X-100, 0.1 mM NADPH, and 0.16 m g / m l NBT. The reaction was terminated by a water wash. Cholinesterase stain was performed by the method of Karnovsky [9]. NDP activity was concentrated at the edges of fibers with a morphology of an endplate, and serial sections stained for cholinesterase confirmed the areas as endplate

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L.L. Kusnet; H.J. Kaminski / Brain Research 730 (1996) 238-242

regions (Fig. 1A,B). NDP activity was also concentrated in the sarcolemmal of certain muscle fibers and was observed in a higher proportion of fibers from EDL than soleus consistent with observations that nNOS is located primarily in fast-twitch fibers [12]. NDP activity was observed at endplates regardless of whether sarcolemmal staining was present. No endplate staining was identified when N A D H was substituted for NADPH. To determine if other oxidative enzymes could account for the reduction of NBT in muscle, crude protein extracts from cerebellum, EDL, and soleus were resolved by nondenaturing polyacrylamide gel electrophoresis. NDP activity was determined as by Kuonen et al. [13]. Single identical bands were found in cerebellum and muscle extracts (data not shown), and the results indicate that all NDP activity in muscle is accounted by a single protein. Other oxidative enzymes may reduce NBT, and therefore, NDP activity is not an absolute confirmation of nNOS expression [1]. We performed double-labeling with nNOS antibodies and o~-bungarotoxin to confirm localization of nNOS to the neuromuscular junction. Sections were prepared in an identical fashion as for NDP histochemistry. Sections were blocked with 1.5% goat serum, incubated

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with anti-nNOS or anti-endothelial NOS (Transduction Laboratories, Lexington, KY), and detected by secondary antibodies of biotinylated anti-rabbit IgG or biotinylated anti-mouse IgG (Vector Laboratories, Burlingam, CA). Texas Red-X conjugated c~-bungarotoxin at 2 i x g / m l (Molecular Probes, Eugene, OR) was included in the secondary antibody incubation to label acetylcholme receptors. The antibodies were detected by a streptavidin fluorescein conjugate (Molecular Probes). Endplates were photographed under rhodamine and fluorescein optics. All EDL and soleus junctions bound anti-nNOS antibodies (Fig. I C,D). nNOS immunostaining of muscle fibers was restricted to the sarcolemma of certain fibers and was similar to the distribution of NDP activity and consistent with a previous report [12]. Endplates of fibers with and without sarcolemmal staining reacted with anti-nNOS antibodies suggesting the endplate localization of nNOS was not related to muscle fiber-type. Sections incubated with antibodies directed against the endothelial isoform of NOS were not stained. To determine if nNOS is localized to the pre- or postsynaptic surface, rats that had undergone right sciatic nerve section were obtained from Zivic-Miller Laborato-

Fig. I. Panel A shows NDP activity concentrated at the edge of an EDL fiber (arrow). Panel B is a serial section stained for cholinesterase activity which confirms the area as an endplate. The majority of the fibers have NDP activity at their periphery. Two fibers without NDP activity are marked and are likely slow-twitch fibers. Panel C demonstrates nNOS immunoreactivity at three soleus muscle endplates, and D shows colocalizati(m with ¢~+bungarotoxin. The scale bar,, are 50 ~m.

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ries (Pittsburgh, PA). Fourteen days after nerve section EDL and soleus muscles of both legs were removed. Denervation was confirmed by the absence of anti-neurofilament antibody staining (anti-neurofilament 160 kDa antibody obtained from Boehringer Mannheim, Indianapolis, IL) of right leg muscles while left leg muscles stained normally (not shown). Muscle fibers of the right leg were atrophic (Fig. 2). nNOS immunoreactivity and NDP activity were present at the neuromuscular junctions (Fig. 2), but the intensity of staining was reduced compared to the normal leg. We conclude that nNOS is concentrated on the postsynaptic surface. No obvious differences in NDP activity or nNOS immunostaining existed between the endplates of the normal leg of denervated animals and of animals which had not undergone denervation. Punctate nNOS immunoreactivity was identified in denervated muscle in a pattern consistent with muscle nuclei (Fig. 2A,B). NDP activity was not evident (Fig. 2C,D). Studies are underway to evaluate the significance of this observation. Extraocular muscle is unique in the presence of fibers which are singly- or multi-innervated. Extraocular muscle is composed primarily of fibers innervated at single en plaque endplates which are similar to endplates of other skeletal muscle. The multi-innervated fibers have several en grappe endplates and some have an additional en plaque

endplate [8,17,20]. As in leg muscle, NDP activity and nNOS immunoreactivity were present at all extraocular muscle en plaque endplates (Fig. 3). nNOS immunoreactivity was found at en grappe endplates, but NDP activity was absent or evident only at rare endplates (Fig. 3). These results cannot be explained by different sensitivities of the methods or concentrations of nNOS since NDP histochemistry is more easily visualized at the endplate than immunostaining. Further, sections contain NDP positive fibers without evidence of endplate staining. The difference in NDP activity at the en grappe endplates compared to en plaque endplates could be caused by (1) an antigenically similar, but enzymatically distinct, isoform of nNOS or (2) variations in local activators of nNOS. The first possibility appears unlikely given the identical electrophoretic mobility of the protein with NDP activity from extraocular muscle and EDL (data not shown). Calcium increases nNOS catalytic activity, and perhaps calcium concentrations differ between en grappe and en plaque endplates. Extraocular muscle en grappe endplates express both the fetal and adult isoforms of the acetylcholine receptor [7], and the acetylcholine receptor density is lower at en grappe endplates [18]. Acetylcholine receptors are permeable to calcium [4,14], and therefore, local calcium concentrations could differ at these endplates. A

Fig. 2. In 14-day denervated muscle, nNOS immunoreactivity (arrow) is concentrated in panel A which colocalizes with c~-bungarotoxin in panel B. Punctate areas of nNOS immunoreactivily are evident throughout the muscle. The staining pattern is consistent with muscle nuclei. Panel C shows concentration of NDP activity at the endplate which in panel D, a serial section stained for cholinesterase activity, is found to be an endplale region. The scale bars are 50 b~m.

L.L. Kusner, H.J. Kaminski / Brain Research 730 (1996J 238 242

lower local calcium concentration at en grappe endplates would lead to decreased activation of nNOS. This difference would be reflected in a lower nNOS catalytic activity and lead to a reduction of NDP activity as reflected by our histochemical results. The lower NDP activity at en grappe endplates may be important in the maintenance of multi-innervation. The identification of nNOS on the postsynaptic surface of the adult neuromuscular junction suggests NO may modulate neuromuscular transmission in adult muscle. NO produced on the postsynaptic surface could diffuse from its site of synthesis across cell membranes to activate target proteins in the nerve terminal. NO may increase or decrease neurotransmitter release from central nervous system neurons, including cholinergic neurons [19]. In X e n o p u s nerve-muscle cultures. S - n i t r o s o - N - a c e t y l p e n i c i l a m i n e , a NO donor, suppresses spontaneous and evoked synaptic currents, and nNOS inhibitors abolish the synaptic suppression induced by postsynaptic depolarization [21]. Innervation has significant affects on muscle protein and gene expression, but the mechanisms which underlie

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this influence are not well understood [5]. Concentrations of nNOS at the endplate would allow NO to act as an integrator of neural activity with postsynaptic messenger systems. Cytosolic calcium concentrations rise in the synaptic region with endplate depolarization [14]. A rise in calcium would activate nNOS and increase NO production [1]. NO could then influence a host of postsynaptic cellular functions through activation of target proteins. Identification of nNOS on the muscle surface of the neuromuscular junction indicates NO may function as a modulator of synaptic transmission and an integrator of neural activity to second messenger systems. This result expands understanding of neuromuscular junction function and may be relevant to the pathogenesis of some neuromuscular diseases, nNOS is bound to the dystrophin membrane complex and is absent from the sarcolemma of dystrophin deficient fibers [2]. Its loss may serve to explain the preferential degeneration of some fast contracting muscle fibers [6,22]. The present investigation suggests that disorders which affect the neuromuscular junction, such as myasthenia gravis and Lambert-Eaton myasthenic syn-

Fig. 3. In extraocular muscle nNOS immunoreactivity in panel A and c,-bungaroloxin in panel B are concentrated at two en plaque endplates (large arro,~s) and multiple en grappe endplates (three are indicated by small arrows). The scale bar is 50 O-re. [n panel C numerous extraocular muscle en grappe endplates are cvident by cholinesterase stain but no corresponding NDP activity is present in panel D. an approximate serial scction, despite numcnm,~ NDP positive fibers. The scale bar is 100 IJ.m.

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L.L. Kusne~; H.J. Kaminski / Brain Research 7:?0 (1996) 238 242

drome,

may

compromise

nNOS

activity.

Drugs,

which

m o d u l a t e n N O S , m a y u l t i m a t e l y p r o v e u s e f u l in t h e treatment of neuromuscular transmission diseases.

Acknowledgements T h i s w o r k is s u p p o r t e d b y N I H G r a n t E Y - 0 0 3 3 2

and

the Department of Veterans Affairs.

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