Elsevier

Nitric Oxide

Volume 44, 30 January 2015, Pages 3-7
Nitric Oxide

Brief Communication
Neuronal NOS localises to human airway cilia

https://doi.org/10.1016/j.niox.2014.11.003Get rights and content

Highlights

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    We sought to localise neuronal NOS (nNOS) in human airway epithelium.

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    nNOS immunoreactivity localised to cilia of healthy bronchial and nasal polyps tissues.

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    nNOS immunoreactivity was specifically blocked by nNOS peptide fragment.

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    nNOS immunoreactivity localised to cilia of healthy human epithelial cells differentiated at an air–liquid interface.

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    nNOS immunofluorescence labelling co-localised with β-tubulin in proximal portion of cilia.

  • •

    We report a novel localisation of nNOS at the proximal portion of cilia in airway epithelium.

Abstract

Background

Airway NO synthase (NOS) isoenzymes are responsible for rapid and localised nitric oxide (NO) production and are expressed in airway epithelium. We sought to determine the localisation of neuronal NOS (nNOS) in airway epithelium due to the paucity of evidence.

Methods and results

Sections of healthy human bronchial tissue in glycol methacrylate resin and human nasal polyps in paraffin wax were immunohistochemically labelled and reproducibly demonstrated nNOS immunoreactivity, particularly at the proximal portion of cilia; this immunoreactivity was blocked by a specific nNOS peptide fragment. Healthy human epithelial cells differentiated at an air–liquid interface (ALI) confirmed the presence of all three NOS isoenzymes by immunofluorescence labelling. Only nNOS immunoreactivity was specific to the ciliary axonemeand co-localised with the cilia marker β-tubulin in the proximal part of the ciliary axoneme.

Conclusions

We report a novel localisation of nNOS at the proximal portion of cilia in airway epithelium and conclude that its independent and local regulation of NO levels is crucial for normal cilia function.

Section snippets

Background

Nitric oxide (NO) is a mobile, reactive and ubiquitous signalling molecule within the airway, regulating vascular and bronchial tone, airway permeability [1] and ciliary beat frequency (CBF) [2], [3], [4], [5]. It is pro-inflammatory with anti-bacterial action [6], [7] and tumouricidal activity [8]. NO is generated from L-arginine by the catalytic activity of nitric oxide synthase (NOS) isoenzymes. The NOS isoenzymes, encoded by NOS1–3 genes on different chromosomes, include neuronal NOS

Ethics statement

Our research received approval from the National Research Ethics Service (NRES), South Central Committee (06/Q1702/109) and locally at the University Hospital Southampton NHS Foundation Trust Research and Development Department (RHMCH10395). All subjects gave written informed consent.

Immunohistochemical labelling of nitric oxide synthases

Human nasal polyp (n = 7 subjects) in paraffin wax sections and healthy human bronchial tissue (n = 3 subjects) in glycol methacrylate (GMA) resin sections were immunostained using standard protocols. For paraffin

nNOS localisation in nasal polyp and healthy bronchial tissues by immunohistochemistry

In nasal polyp tissue sections (n = 7 subjects) and healthy human bronchial tissue sections (n = 3 subjects) nNOS staining was clearly seen in cilia (Fig. 1b and e). The nNOS immunoreactivity was similar to that of cilia specific β-tubulin staining (Fig. 1g) but was evidently absent from the distal third portion of the cilia (Fig. 1b and e). The nNOS immunoreactivity in the proximal portion of the cilia of both the nasal polyp and healthy bronchial tissue was consistently blocked by the

Discussion

We have demonstrated a novel localisation for nNOS consistently to the proximal portion of cilia in human nasal polyp and healthy bronchial tissue, which co-localised with the cilia marker β-tubulin in healthy bronchial tissue sections. The immunolocalisation of nNOS to cilia was confirmed by its consistent abolition using a blocking peptide, and the antibody specificity was corroborated by demonstrating relevant nNOS immunoreactivity in Alzheimer's brain tissue sections. In control experiments

Conclusion

We report a novel localisation of nNOS at the proximal portion of cilia in airway epithelium and conclude that its independent and local regulation of NO levels is crucial for normal cilia development and function.

Source of funding

This research received funding from Wessex Medical Research M06. The National PCD Centre in Southampton is commissioned and funded by NHS England.

Authors' contributions

CLJ designed experiments performed data analysis and wrote the manuscript. JSL and PML contributed to design and manuscript review. WTW, HO and IP performed immunofluorescence and/ or immunohistochemistry of NOS proteins in airway tissue. All authors read and approved the final manuscript.

Acknowledgments

The authors are grateful to all volunteers for their participation in this study. The authors are also grateful to Mr Philip Harries and Mr Salil Nair and their surgical teams for their collection of nasal tissues, to Dr Jane Warner for access to healthy bronchial tissue sections and to Dr Delphine Boche for access to Alzheimer's brain tissue sections. The authors would also like to thank Edith Quinn and staff members of the Histochemistry Research Unit, and Dr David Johnston for Confocal

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