Immunohistochemical co-localization of transient receptor potential vanilloid (TRPV)1 and sensory neuropeptides in the guinea-pig respiratory system

doi:10.1016/j.neuroscience.2006.04.073

Neuroscience

Volume 141, Issue 3, 2006, Pages 1533-1543

https://doi.org/10.1016/j.neuroscience.2006.04.073 Get rights and content

Abstract

Electrophysiological studies within the lung have documented the presence of heterogenous groups of afferent fibers composed of Aδ and C-fibers and studies of somatosensory nerves within the skin reveal a complex pattern of distribution of sensory neuropeptides and transient receptor potential vanilloid (TRPV)1 positive nerves. However, the anatomical location of these different subpopulations of nerves within the lung has not been extensively studied. In the present study we have demonstrated that TRPV1 axons represented only a small proportion of the total number of PGP9.5 staining nerves within guinea-pig tracheal epithelium and only half the number of TRPV1 axons was immunopositive for substance P. In contrast, most TRPV1 positive neurones found within guinea-pig intrapulmonary airways were found to co-localize with sensory neuropeptides substance P and calcitonin gene-related peptide within and beneath the epithelium, around blood vessels, within airway smooth muscle and alveoli, indicative of heterogeneity of TRPV1 positive axons throughout the airways. However, in the smooth muscle layer of the trachea there was evidence of substance P and calcitonin gene-related peptide containing nerves that did not stain for TRPV1. We also demonstrated a complete loss of TRVP1 positive axons in the trachea and intrapulmonary airways and associated loss of bronchoconstriction induced by capsaicin, in animals chronically treated with capsaicin. However, some neuropeptide immunoreactive axons remained in the smooth muscle layer of capsaicin-treated animals which could represent the small subset of neuropeptide containing fibers which do not co-localize with TRPV1. We have provided evidence of heterogeneity of TRPV1 positive nerve fibers, including fibers characterized by lack of co-localization with neuropeptides in various regions of the airways and the existence of neuropeptide containing fibers that were not TRPV1 positive in guinea-pigs.

Section snippets

Animals

Male Dunkin Hartley guinea pigs (300–400 g) were obtained from Charles River Breeding Laboratories (Margate, UK). All animals were housed in our animal facilities for at least 1 week before use and were provided food and water ad libitum. On the day of study, animals were killed by cervical dislocation. All procedures were conducted in accordance with the UK 1986 Animals (Scientific Procedures) Act. Every effort was made to minimize the number of animals used and their suffering.

Tissue preparation

Tissues

TRPV1 and CGRP mapping in guinea-pig airways

Consistent with our previous study (Watanabe et al., 2005), we found evidence of TRPV1 immunoreactive axons throughout the respiratory tract of the guinea pig and in all regions we also observed coexistence with CGRP. Many TRPV1 immunoreactive axons co-localized with CGRP both within the tracheal epithelium and in axons located in a subepithelial plexus (Fig. 1a–f). Similarly, TRPV1 immunoreactive axons co-localized with CGRP within the bronchus (Fig. 1j–l), alveolar tissue (Fig. 1 g–i) and

Discussion

In this study we have mapped the localization and distribution of TRPV1 and of sensory neuropeptides (CGRP, substance P) in guinea-pig extrapulmonary and intrapulmonary airways using immunofluorescence and confocal microscopy. Within the tracheal epithelium, TRPV1 immunoreactive axons were far less abundant than PGP staining ones, and substance P immunoreactive axons were 50% less abundant than the TRPV1 ones. This is consistent with our previous double labeling studies showing that only 12% of

Conclusion

In conclusion, our co-localization studies utilizing confocal microscopy have revealed a number of sub-populations of neuropeptide and TRPV1 expressing afferent nerves within the lung. It is clear that while TRPV1 and substance P/CGRP immunoreactive sensory neurones exist, there is a significant population of TRPV1 immunoreactive nerves which do not co-localize with neuropeptide. The function of these nerves in modulating airway function requires further investigation and highlights the

Acknowledgments

C.P. and D.S. wish to thank Asthma UK and J.V.P. the Wellcome Trust, United Kingdom who supported this study. S.H. was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The authors wish to thank Dr. David Julius for the gift of TRPV1 antibody and Mr. Meng-Chuan Liu for assistance with confocal microscopy.

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It mainly manifests varying degrees of shortness of breath, dyspnea, chest tightness, cough, reversible airflow limitation, prolonged disease, and airway reconstruction caused by recurrence [6,44]. On a global scale, asthma is one of the most prevalent chronic disorders with the morbidity of up to (1.6–36.7) per 10 million [6]. Given the physiopathologic homogeneity, asthma and AR are considered as disorders of the same airway.
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¹: Present address: Division of Respiratory and Infectious Diseases, Department of Internal Medicine, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki City, Kanagawa Prefecture 216-8511, Japan (N. Watanabe); Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Josai International University, 1 Gumyo, Togane, Chiba 283-8555, Japan (S. Horie).

²: Equal first authors.

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Sensory systemImmunohistochemical co-localization of transient receptor potential vanilloid (TRPV)1 and sensory neuropeptides in the guinea-pig respiratory system

Abstract

Section snippets

Animals

Tissue preparation

TRPV1 and CGRP mapping in guinea-pig airways

Discussion

Conclusion

Acknowledgments

Neuroscience

Life Sci

Neurosci Lett

Regul Pept

Brain Res

J Allergy Clin Immunol

Respir Physiol

Neuroscience

Regul Pept

Neurosci Lett

Pain

Lancet

Neuron

J Allergy Clin Immunol

Neurosci Lett

Brain Res

Neuron

Neuroscience

Pulm Pharmacol Ther

Immunocytochemical localization of trkA receptors in chemically identified subgroups of adult rat sensory neurons

Eur J Neurosci

Substance P-immunoreactive sensory axons in the rat respiratory tracta quantitative study of their distribution and role in neurogenic inflammation

J Comp Neurol

Intraepithelial vagal sensory nerve terminals in rat pulmonary neuroepithelial bodies express P2X(3) receptors

Am J Respir Cell Mol Biol

Dual sensory innervation of pulmonary neuroepithelial bodies

Am J Respir Cell Mol Biol

Localization of cholinergic nerves in lower airways of guinea pigs using antisera to choline acetyltransferase

Am J Physiol

The capsaicin receptora heat-activated ion channel in the pain pathway

Nature

TRP channels as cellular sensors

Nature

Sensory system
Immunohistochemical co-localization of transient receptor potential vanilloid (TRPV)1 and sensory neuropeptides in the guinea-pig respiratory system