TRPA1 is a major oxidant sensor in murine airway sensory neurons
Bret F. Bessac, Michael Sivula, Christian A. von Hehn, Jasmine Escalera, Lauren Cohn, Sven-Eric Jordt
Bret F. Bessac, Michael Sivula, Christian A. von Hehn, Jasmine Escalera, Lauren Cohn, Sven-Eric Jordt
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Research Article

TRPA1 is a major oxidant sensor in murine airway sensory neurons

Abstract

Sensory neurons in the airways are finely tuned to respond to reactive chemicals threatening airway function and integrity. Nasal trigeminal nerve endings are particularly sensitive to oxidants formed in polluted air and during oxidative stress as well as to chlorine, which is frequently released in industrial and domestic accidents. Oxidant activation of airway neurons induces respiratory depression, nasal obstruction, sneezing, cough, and pain. While normally protective, chemosensory airway reflexes can provoke severe complications in patients affected by inflammatory airway conditions like rhinitis and asthma. Here, we showed that both hypochlorite, the oxidizing mediator of chlorine, and hydrogen peroxide, a reactive oxygen species, activated Ca2+ influx and membrane currents in an oxidant-sensitive subpopulation of chemosensory neurons. These responses were absent in neurons from mice lacking TRPA1, an ion channel of the transient receptor potential (TRP) gene family. TRPA1 channels were strongly activated by hypochlorite and hydrogen peroxide in primary sensory neurons and heterologous cells. In tests of respiratory function, Trpa1–/– mice displayed profound deficiencies in hypochlorite- and hydrogen peroxide–induced respiratory depression as well as decreased oxidant-induced pain behavior. Our results indicate that TRPA1 is an oxidant sensor in sensory neurons, initiating neuronal excitation and subsequent physiological responses in vitro and in vivo.

Authors

Bret F. Bessac, Michael Sivula, Christian A. von Hehn, Jasmine Escalera, Lauren Cohn, Sven-Eric Jordt

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Figure 4

H2O2 induces TRPA1-dependent influx of Ca2+ and ionic currents in mustard oil–responsive sensory neurons.

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Activation of heterologously expressed TRPA1 channels by H2O2. (A) R...
(A) Responses of cultured DRG neurons from littermate Trpa1+/+ and Trpa1–/– mice to 5 mM H2O2, followed by 3 μM capsaicin, as measured by Fura-2 imaging. Trpa1–/– neurons showed no [Ca2+]i increase after H2O2 exposure, but were activated by capsaicin. Pseudocolors denote 0–3 μM [Ca2+]i. (B) Activation of Ca2+ influx by H2O2 into DRG neurons plotted against time. Average [Ca2+]i concentration of neurons activated by application of H2O2 followed by mustard oil, capsaicin, and 65 mM KCl. Thick and thin lines denote mean and ± SEM, respectively. Neurons (n = 189 [Trpa1+/+]; 146 [Trpa1–/–]) were analyzed at ×10 magnification. (C) Activation of DRG neurons (n = 161) by 5 mM H2O2, NaOCl (24 ppm), 100 μM mustard oil, 5 μM capsaicin, and 65 mM KCl. Neurons were considered activated when [Ca2+]i exceeded 500 nM. Values denote activated KCl-sensitive cells. (D) Activation of DRG neurons (n = 130) by NaOCl (24 ppm), 5 mM H2O2, 100 μM mustard oil, 5 μM capsaicin, and 65 mM KCl. (E) Kinetics of H2O2-activated cationic currents and ruthenium red–induced block in a cultured murine sensory neuron. H2O2 was superfused after 50-s initiation of whole-cell configuration, after which ruthenium red was coapplied at 210 s. Currents were measured using a ±80 mV voltage ramp protocol over 100 ms at 0.5-Hz intervals (0 mV holding potential throughout). Intracellular Cs-based solution contained 10 mM EGTA. (F) Representative current-voltage relationships of currents recorded from a DRG neuron before application of H2O2 (black), during maximal activation by H2O2 (green), and after application of 20 μM ruthenium red (red). Currents were measured as in C. Error bars represent SEM.