Cited by 96Nitric Oxide and Endothelin EffectsCardiovascular and exercise physiologyMuscle Physiology and Disorders

Real‐time measurement of nitric oxide in single mature mouse skeletal muscle fibres during contractions

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Pye, Deborah, Palomero, Jesus, Kabayo, Tabitha, Jackson, Malcolm J.·2007·The Journal of Physiology

Abstract

Nitric oxide (NO) is thought to play multiple roles in skeletal muscle including regulation of some adaptations to contractile activity, but appropriate methods for the analysis of intracellular NO activity are lacking. In this study we have examined the intracellular generation of NO in isolated single mature mouse skeletal muscle fibres at rest and following a period of contractile activity. Muscle fibres were isolated from the flexor digitorum brevis muscle of mice and intracellular NO production was visualized in real‐time using the fluorescent NO probe 4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate (DAF‐FM DA). Some leakage of DAF‐FM was apparent from fibres loaded with the probe, but they retained sufficient probe to respond to changes in intracellular NO following addition of the NO donor 3‐(2‐hydroxy‐1‐methyl‐2‐nitrosohydrazino)‐N‐methyl‐1‐propanamine (NOC‐7) up to 30 min after loading. Electrically stimulated contractions in isolated fibres increased the rate of change in DAF‐FM fluorescence by ∼48% compared to non‐stimulated fibres (P < 0.05) and the rate of change in DAF‐FM fluorescence in the stimulated fibres returned to control values by 5 min after contractions. Treatment of isolated fibres with the NO synthase inhibitors NG‐nitro‐l‐arginine methyl ester hydrochloride (l‐NAME) or NG‐monomethyl‐l‐arginine (l‐NMMA) reduced the increase in DAF‐FM fluorescence observed in response to contractions of untreated fibres. Treatment of fibres with the cell‐permeable superoxide scavenger 4,5‐dihydroxy‐1,3‐benzenedisulphonic acid (Tiron) also reduced the increase in fluorescence observed during contractions suggesting that superoxide, or more probably peroxynitrite, contributes to the fluorescence observed. Thus this technique can be used to examine NO generation in quiescent and contracting skeletal muscle fibres in real time, although peroxynitrite and other reactive nitrogen species may potentially contribute to the fluorescence values observed.

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Crossref · 10.1113/jphysiol.2006.125930

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Authors
Pye, D., Palomero, J., Kabayo, T., & Jackson, M. J.
Affiliations
University of Liverpool (GB)
Venue
The Journal of Physiology
Publisher
Wiley
Published
2007-05-04
Volume
581
Issue
1
Pages
309-318
ISSN
  • 0022-3751 (ISSN-L)
  • 1469-7793
OpenAlex
W1586255609
Cited by
96
References
39
Citations by year
2026: 1 · 2025: 2 · 2024: 3 · 2023: 3 · 2022: 5 · 2021: 6 · 2020: 9 · 2019: 3 · 2018: 5 · 2017: 4 · 2016: 5 · 2015: 4 · 2014: 5 · 2013: 11 · 2012: 5
FWCI
3.12
Type
journal article
Language
en
Open access
Locations
  • The Journal of Physiology (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • Hispana (publishedVersion, cc-by-nc-nd)
  • Gredos (University of Salamanca) (submittedVersion, cc-by-nc-nd)
Funders
Wellcome Trust (073263 ⁄ Z ⁄ 03)
Topics
  • Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
  • Health Sciences › Medicine › Complementary and alternative medicine › Cardiovascular and exercise physiology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
Keywords
nitric oxide, skeletal muscle fibers, muscle contraction, NO synthase inhibition, superoxide, peroxynitrite, reactive nitrogen species, flexor digitorum brevis, L-NAME, L-NMMA, Tiron
MeSH terms
Animals, Cells, Cultured, Electric Stimulation, Enzyme Inhibitors, Female, Fluoresceins, Indicators and Reagents, Mice, Inbred C57BL, Microscopy, Fluorescence, Muscle Contraction, Nitric Oxide, 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt, Muscle, Skeletal, Muscle Fibers, Skeletal, Nitric Oxide Synthase, omega-N-Methylarginine, NG-Nitroarginine Methyl Ester, Mice

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