Nitric Oxide in Skeletal Muscle: Inhibition of Nitric Oxide Synthase Inhibits Walking Speed in Rats

doi:10.1006/niox.2001.0348

Nitric Oxide

Volume 5, Issue 3, June 2001, Pages 219-232

https://doi.org/10.1006/niox.2001.0348 Get rights and content

Abstract

Nitric oxide (NO·) is a multifunctional messenger molecule generated by a family of enzymes called the nitric oxide synthases (NOSs). Although NOSs have been identified in skeletal muscle, specifically brain NOS (bNOS) and endothelial NOS (eNOS), their role has not been well clarified. The goals of this investigation were to (1) characterize the immunoreactivity, Ca²⁺ dependence, and activity of NOS in human and rat skeletal muscle and (2) using a rat model, investigate the effect of chronic blockade of NOS on skeletal muscle structure and function. Our results showed that both human and rodent skeletal muscle had NOS activity. This NOS activity was similar to that of the endothelial and brain NOS isoforms in that it was calcium-dependent. However, Western blot analysis consistently showed that a polyclonal antibody raised against a peptide sequence of human inducible NOS (iNOS) reacted with a protein with a molecular weight (95 kDa) that was different from that of other NOS isoforms. RT-PCR analysis identified the mRNA expression of not only eNOS and bNOS but also iNOS in human and rat muscle. Inhibition of nitric oxide synthase in rats with N^ω-nitro- $l$ -arginine methyl ester ( $l$ -NAME) resulted in a progressive, severe reduction in walking speed (30-fold reduction in walking velocity at day 22, P < 0.001), muscle fiber cross-sectional area (40% reduction at day 22, P < 0.001), and muscle mass (40% reduction in dry weight at day 22, P < 0.01). Rats fed the same regimen of the enantiomer of $l$ -NAME ( $d$ -NAME) had normal motor function, muscle fiber morphology, and muscle mass. Taken together, these results imply that there may be a novel nitric oxide synthase in muscle and that NO· generated from muscle may be important in muscle function.

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Perhaps the concentration of 2 mM/L Arg, chosen for its ability to stimulate in vitro the activation of mice satellite cells (Betters et al., 2008), was not sufficient to induce changes in vivo on the myogenic markers studied, or the duration of Arg treatment too short. We used embryonic L-NAME bath in order to determine if treatment of trout eggs with a NOS inhibitor would act on the fate of muscle precursor cells and on muscle cellularity, as it occurs in vivo in mice and rats injected intraperitoneally with L-NAME or fed with L-NAME in drinking water and in chick injected in ovo by NPLA (another NOS inhibitor) (Anderson, 2000; Carrazo et al., 2014; Tatsumi et al., 2006; Wang et al., 2001). We chose to use a low concentration of L-NAME (1 mM/L) as we did not want to alter embryo survival and this aim was attained, confirming previous results (Eddy et al., 1999), but this treatment did not alter neither the embryonic pattern of expression of myf5 and fmhc nor the number of white muscle fibres formed at hatching.
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Chronic exercise training causes an upregulation of nNOS and increased enzyme activity in the diaphragm (Javeshghani et al., 2000). Chronic NOS blockade inhibits skeletal muscle adaptation to chronic endurance exercise and results in severe loss of walking speed in rats (Wang et al., 2001). nNOS−/− mice show decreased muscle endurance (Percival et al., 2008) and when nNOS knockouts are challenged with LPS, diaphragm dysfunction is exacerbated compared to control (Comtois et al., 2001).
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Chronic nNOS blockade did not alter sternohyoid muscle peak force or force–frequency relationship in sham or CIH-treated animals. In contrast, chronic nNOS blockade significantly decreased sternohyoid muscle endurance with equivalent effects in sham and CIH-treated rats.
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The induction of mechanical damage to gastrocnemius muscle has been shown to result in increased NO formation, which is believed to initiate a signaling process for damage repair [88]. In addition, the importance of NO to muscle function, has been demonstrated as NO inhibition resulted in severe reduction in walking speed of rats [89]. Therefore, one can suggest that NO, which is generated during muscle contraction as a result of nNO [29], eNO [90–92] and iNO [93,94] activation, could cause decreased force generation, pain as a protective mechanism but also obligatory to the repair process by satellite cell activation, proliferation, and fusion by the activating follistatin (Fig. 2).
Skeletal muscle hosts all of the isoforms of nitric oxide synthase (NOS). It is well documented that nitric oxide (NO) regulates force generation and satellite cell activation, and therefore, damage repair of skeletal muscle. NO can also activate nociceptors of C-fibers, thereby causing the sensation of pain. Although delayed-onset of muscle soreness (DOMS) is associated with decreased maximal force generation, pain sensation and sarcomere damage, there is a paucity of research linking NO and DOMS. The present mini-review attempts to elucidate the possible relationship between NO and DOMS, based upon current literature.
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Skeletal muscle is a target organ for a variety of chemicals. Adverse or toxic effects on skeletal muscles can range from minor muscle weakness or slight pain to complete paralysis. Next to the brain, skeletal muscles are major targets for the toxicity of organophosphate (OP) nerve agents. Morbidity and mortality associated with OP intoxication is due to the effects of these compounds on skeletal muscles in general and muscles of respiration in particular. Deaths from overdose of OPs are due in part to respiratory paralysis by depolarizing neuromuscular blockade. Exposure to sublethal signs-producing doses of an OP nerve agent exerts prominent motor, behavioral, and autonomic symptoms. The motor symptoms are fasciculations, fibrillations, and body tremors. Fasciculations and fibrillations are due to antidromic neural discharge from excess junctional ACh, while tremors are of a central origin. Various pharmacologic and therapeutic drugs have been tested to prevent or treat myopathy. Drugs effective in treatment of neuromuscular signs of anti-ChE toxicity include: oximes that reactivate the phosphorylated AChE, subparalyzing as well as paralyzing doses of d-tubocurarine, atropine sulfate/atropine methyl nitrate, diazepam, and creatine phosphate.
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Aging is associated with progressive decline of skeletal muscle mass and function. This condition, termed sarcopenia, is associated with several adverse outcomes, including loss of autonomy and mortality. Due to the high prevalence of sarcopenia, a deeper understanding of its pathophysiology and possible remedies represents a high public health priority. Evidence suggests the existence of a relationship between declining growth hormone (GH) and insulin-like growth factor-1 (IGF-1) levels and age-related changes in body composition and physical function. Therefore, the age-dependent decline of GH and IGF-1 serum levels may promote frailty by contributing to the loss of muscle mass and strength. Preclinical studies showed that infusion of angiotensin II produced a marked reduction in body weight, accompanied by decreased serum and muscle levels of IGF-1. Conversely, overexpression of muscle-specific isoform of IGF-1 mitigates angiotensin II-induced muscle loss. Moreover, IGF-1 serum levels have been shown to increase following angiotensin converting enzyme inhibitors (ACEIs) treatment. Here we will review the most recent evidence regarding age-related changes of the GH/IGF-1 axis and its modulation by several interventions, including ACEIs which might represent a potential novel strategy to delay the onset and impede the progression of sarcopenia.
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In this study, nitric oxide synthase immunohistochemistry supported by nicotinamide adenine dinucleotide phosphate diaphorase histochemistry was used to demonstrate the nitric oxide synthase immunoreactivity in the monosynaptic Ia-motoneuron pathway exemplified by structural components of the afferent limb of the soleus H-reflex in the dog. A noticeable number of medium-sized intensely nitric oxide synthase immunoreactive somata (1000–2000 μm² square area) and large intraganglionic nitric oxide synthase immunoreactive fibers, presumed to be Ia axons, was found in the L7 and S1 dorsal root ganglia. The existence of nitric oxide synthase immunoreactive fibers (6–8 μm in diameter, not counting the myelin sheath) was confirmed in L7 and S1 dorsal roots and in the medial bundle of both dorsal roots before entering the dorsal root entry zone. By virtue of the funicular organization of nitric oxide synthase immunoreactive fibers in the dorsal funiculus, the largest nitric oxide synthase immunoreactive fibers represent stem Ia axons located in the deep portion of the dorsal funiculus close to the dorsomedial margin of the dorsal horn. Upon entering the gray matter of L7 and S1 segments and passing through the medial half of the dorsal horn, tapered nitric oxide synthase immunoreactive collaterals of the stem Ia fibers pass through the deep layers of the dorsal horn and intermediate zone, and terminate in the group of homonymous motoneurons in L7 and S1 segments innervating the gastrocnemius-soleus muscles. Terminal fibers issued in the ventral horn intensely nitric oxide synthase immunoreactive terminals with long axis ranging from 0.7 to ≥15.1 μm presumed to be Ia bNOS-IR boutons. This finding is unique in that it focuses directly on nitric oxide synthase immunopositivity in the signalling transmitted by proprioceptive Ia fibers. Nitric oxide synthase immunoreactive boutons were found in the neuropil of Clarke’s column of L4 segment, varying greatly in size from 0.7 to ≥15.1 μm in length × 0.7 to 4.8 μm wide. Subsequent to identification of the afferent nitric oxide synthase immunoreactive limb of the monosynaptic Ia-motoneuron pathway on control sections, intramuscular injections of the retrograde tracer Fluorogold into the gastrocnemius-soleus muscles, combined with nitric oxide synthase immunohistochemistry of L7 and S1 dorsal root ganglia, confirmed the existence of a number of medium-sized nitric oxide synthase immunoreactive somata (1000–2000 μm² square area) in the dorsolateral part of both dorsal root ganglia, presumed to be proprioceptive Ia neurons. Concurrently, large nitric oxide synthase immunoreactive fibers were detected at the input and output side of both dorsal root ganglia. S1 and S2 dorsal rhizotomy caused a marked depletion of nitric oxide synthase immunoreactivity in the medial bundle of S1 and S2 dorsal roots and in the dorsal funiculus of S1, S2 and lower lumbar segments. In addition, anterograde degeneration of large nitric oxide synthase immunoreactive Ia fibers in the dorsal funiculus of L7-S2 segments produces direct evidence that the afferent limb of the soleus H-reflex is nitric oxide synthase immunoreactive and presents new immunohistochemical characteristics of the monosynaptic Ia-motoneuron pathway, unseparably coupled with the performance of the stretch reflex.

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¹: To whom correspondence should be addressed at Department of Orthopaedic Surgery, The St George Hospital, Kogarah, Sydney, NSW 2217, Australia. Fax: 61-2-93503967. E-mail: [email protected].

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Regular ArticleNitric Oxide in Skeletal Muscle: Inhibition of Nitric Oxide Synthase Inhibits Walking Speed in Rats

Abstract

J. Biol. Chem.

J. Biol. Chem.

Life Sci.

Biophys. Res. Commun.

FEBS Lett.

FEBS Lett.

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Cell

Biochem. Biophys. Res. Commun.

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Biochem. Biophys. Res. Commun.

Endothelial nitric oxide synthase: Molecular cloning and characterization of a distinct constitutive enzyme isoform

Proc. Natl. Acad. Sci. USA

Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase

Nature

Cloning and characterization of inducible nitric oxide synthase from mouse macrophages

Science

Calmodulin is a subunit of nitric oxide synthase from macrophages

J. Exp. Med.

Nitric oxide: An important articular free radical

J. Bone Joint Surgery (Am)

Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharide

J. Immunol.

Induction of calcium-dependent nitric oxide synthases by sex hormones

Proc. Natl. Acad. Sci. USA

Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter

Nature

Formation of nitric oxide from L-arginine in the central nervous system: A transduction mechanism for stimulation of the soluble guanylate cyclase

Proc. Natl. Acad. Sci. USA

Regular Article
Nitric Oxide in Skeletal Muscle: Inhibition of Nitric Oxide Synthase Inhibits Walking Speed in Rats