Real‐time measurement of nitric oxide in single mature mouse skeletal muscle fibres during contractions
Open accessAbstract
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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Mitochondria are not responsible for ROS generation during exercise.
“However, mitochondria are not responsible for ROS generation during exercise (20–22).”
From Skeletal muscle NOX4 is required for adaptive responses that prevent insulin resistance (2021) · cites this paper as [20]
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Ulanova, A. D., Gritsyna, Y., Salmov, N. N., Lomonosova, Y. N., Belova, S. P., Nemirovskaya, T. L., Shenkman, B. S., & Vikhlyantsev, I. M. (2019) · Frontiers in Physiology · doi:10.3389/fphys.2019.01221 · Cited by 16 · see citing works
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Redox Homeostasis in Age-Related Muscle Atrophy
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Mechanistic models to guide redox investigations and interventions in musculoskeletal ageing
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Self-citationChemical Cytometry Quantitates Superoxide Levels in the Mitochondrial Matrix of Single Myoblasts
Xu, X. M., & Arriaga, E. A. (2010) · Analytical Chemistry · doi:10.1021/ac101509d · Cited by 11 · see citing works
The Positive and Negative Aspects of Reactive Oxygen Species in Sports Performance
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Heaton, R. A., Ball, S. T. M., Staunton, C. A., Mouly, V., Jones, S. W., McArdle, A., & Jackson, M. J. (2024) · Free Radical Biology and Medicine · doi:10.1016/j.freeradbiomed.2024.11.053 · Cited by 9 · see citing works
Self-citationLomonosova, Y. N., Kalamkarov, G. R., Бугрова, А. Е., Shevchenko, T. F., Kartashkina, N. L., Lysenko, E. A., Shenkman, E. A., & Nemirovskaya, T. L. (2012) · Biochemistry (Moscow) · doi:10.1134/s0006297912020137 · Cited by 9 · see citing works
Fernández-Puente, E., Martín-Prieto, E., Márquez, C. M., & Palomero, J. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms24098082 · Cited by 7 · see citing works
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Metabolic Factors in Skeletal Muscle Fatigue
Place, N., & Westerblad, H. (2022) · Physiology in health and disease · doi:10.1007/978-3-030-94305-9_17 · Cited by 6 · see citing works
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Self-citationJackson, M. J. (2025) · Sports Medicine and Health Science · doi:10.1016/j.smhs.2025.02.002 · Cited by 5 · see citing works
Self-citationGong, H., Liu, L., Su, W., Zhu, Z., Liu, Y., Lian, Y., Wei, P., Cao, Z., Zhang, T., & Jiang, C.-L. (2019) · The Journal of Sports Medicine and Physical Fitness · doi:10.23736/s0022-4707.18.08456-6 · Cited by 4 · see citing works
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Self-citationProtective effect of nitric oxide on cytoskeletal proteins in rat soleus under eccentric exercise
Lomonosova, Y. N., Zheleznyakova, A. V., Бугрова, А. Е., Zhiryakova, A. V., Kalamkarov, G. R., & Nemirovskaya, T. L. (2009) · BIOPHYSICS · doi:10.1134/s0006350909030191 · Cited by 3 · see citing works
Aging and Exercise-Induced Reactive Oxygen Species
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Jackson, M. J. (2013) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/b978-0-12-405881-1.00017-3 · Cited by 2 · see citing works
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Blum, K., Han, D. K. M., Oscar‐Berman, M., Reinl, G., DiNubile, N. A., Madigan, M. A., Bajaj, A., Downs, B. W., Giordano, J., Westcott, W., et al. (2013) · Health · doi:10.4236/health.2013.511a1004 · Cited by 1 · see citing works
Signaling effects of substrate stimulation of nNOS in rat soleus after eccentric exercise
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Jackson, M. J. (2024) · Proceedings of The Nutrition Society · doi:10.1017/s0029665124004877 · Cited by 1 · see citing works
Self-citationGusnanto, A., Earl, K., Sakellariou, G. K., Owens, D., Lightfoot, A., Fawcett, S. A., Owen, E. M., Staunton, C. A., Shu, T., Croden, F., et al. (2020) · medRxiv · doi:10.1101/2020.08.17.20164715 · Cited by 1 · see citing works
Self-citationRegulation of Skeletal Muscle Glucose Uptake: A Focus on Nitric Oxide Synthase and Rac1 Signalling
Kerris, J. P. (2020) · Victoria University Research Repository (Victoria University)
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Self-citationJammes, Y. (2013) · Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature · doi:10.3410/f.717956105.793473214
Decoding S-nitrosylation: Theoretical aspects and analytical approaches
Matrullo, G., Pecorari, C., & Filomeni, G. (2024) · Elsevier eBooks · doi:10.1016/b978-0-443-22354-9.00003-6
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Self-citation
- Authors
- Pye, D., Palomero, J., Kabayo, T., & Jackson, M. J.
- Affiliations
- University of Liverpool (GB)
- ORCID
- Jackson, Malcolm J.
- 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
- PMC
- PMC2075220
- 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
- green
- www.ncbi.nlm.nih.gov
- 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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