Publications

Inflammation can increase the excitability of bronchopulmonary C-fibers leading to excessive sensations and reflexes (e.g. wheeze and cough). We have previously shown modulation of peripheral nerve terminal mitochondria by antimycin A causes hyperexcitability in TRPV1-expressing bronchopulmonary C-fibers through the activation of protein kinase C (PKC). Here, we have investigated the PKC isoform responsible for this signaling. We found PKCβ1, PKCδ and PKCε were expressed by many vagal neurons, with PKCα and PKCβ2 expressed by subsets of vagal neurons. In dissociated vagal neurons, antimycin A caused translocation of PKCα but not the other isoforms, and only in TRPV1-lineage neurons. In bronchopulmonary C-fiber recordings, antimycin A increased the number of action potentials evoked by α,β-methylene ATP. Selective inhibition of PKCα, PKCβ1 and PKCβ2 with 50 nM bisindolylmaleimide I prevented the antimycin-induced bronchopulmonary C-fiber hyperexcitability, whereas selective inhibition of only PKCβ1 and PKCβ2 with 50 nM LY333531 had no effect. We therefore conclude that PKCα is required for antimycin-induced increases in bronchopulmonary C-fiber excitability.

Air pollutants pose a serious worldwide health hazard, causing respiratory and cardiovascular morbidity and mortality. Pollutants perturb the autonomic nervous system, whose function is critical to cardiopulmonary homeostasis. Recent studies suggest that pollutants can stimulate defensive sensory nerves within the cardiopulmonary system, thus providing a possible mechanism for pollutant-induced autonomic dysfunction. A better understanding of the mechanisms involved would likely improve the management and treatment of pollution-related disease.

The ATP-sensitive P2X(2) ionotropic receptor plays a critical role in a number of signal processes including taste and hearing, carotid body detection of hypoxia, the exercise pressor reflex and sensory transduction of mechanical stimuli in the airways and bladder. Elucidation of the role of P2X(2) has been hindered by the lack of selective tools. In particular, detection of P2X(2) using established pharmacological and biochemical techniques yields dramatically different expression patterns, particularly in the peripheral and central nervous systems. Here, we have developed a knockin P2X(2)-cre mouse, which we crossed with a cre-sensitive tdTomato reporter mouse to determine P2X(2) expression. P2X(2) was found in more than 80% of nodose vagal afferent neurons, but not in jugular vagal afferent neurons. Reporter expression correlated in vagal neurons with sensitivity to αβmATP. P2X(2) was expressed in 75% of petrosal afferents, but only 12% and 4% of dorsal root ganglia and trigeminal afferents, respectively. P2X(2) expression was limited to very few cell types systemically. Together with the central terminals of P2X(2)-expressing afferents, reporter expression in the CNS was mainly found in brainstem neurons projecting mossy fibers to the cerebellum, with little expression in the hippocampus or cortex. The structure of peripheral terminals of P2X(2)-expressing afferents was demonstrated in the tongue (taste buds), carotid body, trachea and esophagus. P2X(2) was observed in hair cells and support cells in the cochlear, but not in spiral afferent neurons. This mouse strain provides a novel approach to the identification and manipulation of P2X(2)-expressing cell types.Significance statement Inhibitor and knockout studies have demonstrated the critical role of P2X(2) in multiple sensory signaling pathways. Nevertheless, P2X(2) expression patterns are controversial, as biochemical studies suggest widespread expression whereas functional studies suggest restricted expression. Functional characterization is further complicated by heteromeric P2X(2/3) channels that have hybrid pharmacology and biophysical properties. We have developed a P2X(2)-cre mouse to determine the expression pattern of P2X(2) In the periphery, P2X(2) expression is found in almost all nodose sensory afferents but is limited to only minor subsets of trigeminal and DRG afferents. Centrally, P2X(2) is mostly expressed in neurons projecting mossy fibers to the cerebellum. Thus we provide novel evidence for the specific expression of P2X(2), which is more limited than previously thought.

Vagal afferent sensory nerves, originating in jugular and nodose ganglia, are comprised of functionally distinct subsets whose activation evokes distinct thoracic and abdominal reflex responses. We used Cre-expressing mouse strains to identify specific vagal afferent populations and map their central projections within the brainstem. We show that Pirt is expressed in virtually all vagal afferents; whereas 5HT3 is expressed only in nodose neurons, with little expression in jugular neurons. TRPV1, the capsaicin receptor, is expressed in a subset of small nodose and jugular neurons. Tac1, the gene for tachykinins, is expressed predominantly in jugular neurons, some of which also express TRPV1. Vagal fibers project centrally to the nucleus tractus solitarius (nTS), paratrigeminal complex, area postrema and to a limited extent the dorsal motor nucleus of the vagus. nTS subnuclei preferentially receive projections by specific afferent subsets, with TRPV1+ fibers terminating in medial and dorsal regions predominantly caudal of obex, whereas TRPV1-negative fibers terminate in ventral and lateral regions throughout the rostral-caudal aspect of the medulla. Many vagal Tac1+ afferents (mostly derived from the jugular ganglion) terminate in the nTS. The paratrigeminal complex was the target of multiple vagal afferent subsets. Importantly, lung-specific TRPV1+ and Tac1+ afferent terminations were restricted to the caudal medial nTS, with no innervation of other medulla regions. In summary, this study identifies the specific medulla regions innervated by vagal afferent subsets. The distinct terminations provide a neuroanatomic substrate for the diverse range of reflexes initiated by vagal afferent activation.

Inflammation causes activation of nociceptive sensory nerves, resulting in debilitating sensations and reflexes. Inflammation also induces mitochondrial dysfunction through multiple mechanisms. Sensory nerve terminals are densely packed with mitochondria, suggesting that mitochondrial signaling may play a role in inflammation-induced nociception. We have previously shown that agents that induce mitochondrial dysfunction, such as antimycin A, activate a subset of nociceptive vagal sensory nerves that express transient receptor potential (TRP) channels ankyrin 1 (A1) and vanilloid 1 (V1). However, the mechanisms underlying these responses are incompletely understood. Here, we studied the contribution of TRPA1, TRPV1 and reactive oxygen species (ROS) to antimycin A-induced vagal sensory nerve activation in dissociated neurons and at the sensory terminals of bronchopulmonary C-fibers. Nociceptive neurons were defined chemically and genetically. Antimycin A-evoked activation of vagal nociceptors in a Fura2 Ca(2+) assay correlated with TRPV1 responses compared to TRPA1 responses. Nociceptor activation was dependent on both TRP channels, with TRPV1 predominating in a majority of responding nociceptors and TRPA1 predominating only in nociceptors with the greatest responses. Surprisingly, both TRPA1 and TRPV1 were activated by H2O2 when expressed in HEK293. Nevertheless, targeting ROS had no effect of antimycin A-evoked TRPV1 activation in either HEK293 or vagal neurons. In contrast, targeting ROS inhibited antimycin A-evoked TRPA1 activation in HEK293, vagal neurons and bronchopulmonary C-fibers, and a ROS-insensitive TRPA1 mutant was completely insensitive to antimycin A. We therefore conclude that mitochondrial dysfunction activates vagal nociceptors by ROS-dependent (TRPA1) and ROS-independent (TRPV1) mechanisms.

KEY POINTS: We investigated the cardiovascular and respiratory responses of the normotensive Wistar-Kyoto (WKY) rat and the Spontaneously Hypertensive (SH) rat to inhalation and intravenous injection of the noxious stimuli allyl isothiocyanate (AITC). AITC inhalation evoked atropine-sensitive bradycardia in conscious WKY, and evoked atropine-sensitive bradycardia and atenolol-sensitive tachycardia with premature ventricular contractions (PVCs) in conscious SH. Intravenous injection of AITC evoked bradycardia but no tachycardia/PVCs in conscious SH. Inhalation and injection of AITC causes similar bradypnea in conscious SH and WKY. Anesthesia (inhaled isoflurane) inhibited the cardiac reflexes evoked by inhaled AITC but not injected AITC. Data indicates the presence of a de novo nociceptive pulmonary-cardiac reflex triggering sympathoexcitation in the SH, and this reflex is dependent on vagal afferents but is not due to steady state blood pressure or due to remodeling of vagal efferent function. ABSTRACT: Inhalation of noxious irritants/pollutants activates airway nociceptive afferents resulting in reflex bradycardia in healthy animals. Nevertheless, noxious pollutants evoke sympathoexcitation (tachycardia, hypertension) in cardiovascular disease patients. We hypothesize that cardiovascular disease alters nociceptive pulmonary-cardiac reflexes. Here, we studied reflex responses to irritants in normotensive Wistar-Kyoto rats (WKY) and Spontaneously Hypertensive rats (SH). Inhaled allyl isothiocyanate (AITC) evoked atropine-sensitive bradycardia with atrial-ventricular (AV) block in conscious WKY, thus indicating a parasympathetic reflex. Conversely, inhaled AITC in conscious SH evoked complex brady-tachycardia with both AV block and premature ventricular contractions (PVCs). Atropine abolished the bradycardia and AV block, but the atropine-insensitive tachycardia and PVCs were abolished by the beta1 adrenoceptor antagonist atenolol. The aberrant AITC-evoked reflex in SH was not reduced by acute blood pressure reduction by captopril. Surprisingly, intravenous AITC only evoked bradycardia in conscious SH and WKY. Furthermore, anesthesia reduced the cardiac reflexes evoked by inhaled but not injected AITC. Nevertheless, anesthesia had little effect on AITC-evoked respiratory reflexes. Such data suggest distinct differences in nociceptive reflex pathways dependent on cardiovascular disease, administration route and downstream effector. AITC-evoked tachycardia in decerebrate SH was abolished by vagotomy. Finally, there was no difference in the cardiac responses of WKY and SH to vagal efferent electrical stimulation. Our data suggest that AITC inhalation in SH evokes de novo adrenergic reflexes following vagal afferent activation. This aberrant reflex is independent of steady state hypertension and is not evoked by intravenous AITC. We conclude that preexisting hypertension aberrantly shifts nociceptive pulmonary-cardiac reflexes towards sympathoexcitation. This article is protected by copyright. All rights reserved.

Diesel exhaust particulate (DEP) causes pulmonary irritation and inflammation, which can exacerbate asthma and other diseases. These effects may arise from the activation of transient receptor potential ankyrin-1 (TRPA1). This study shows that a representative DEP can activate TRPA1-expressing pulmonary C-fibers in the mouse lung. Furthermore, DEP collected from idling vehicles at an emissions inspection station, the tailpipe of an on-road "black smoker" diesel truck, waste DEP from a diesel exhaust filter regeneration machine, and NIST SRM 2975 can activate human TRPA1 in lung epithelial cells to elicit different biological responses. The potency of the DEP, particle extracts, and selected chemical components was compared in TRPA1 over-expressing HEK-293 and human lung cells using calcium flux and other toxicologically relevant end-point assays. Emission station DEP was the most potent and filter DEP the least. Potency was related to the percentage of ethanol extractable TRPA1 agonists and was equivalent when equal amounts of extract mass was used for treatment. The DEP samples were further compared using scanning electron microscopy, energy-dispersive X-ray spectroscopy, gas chromatography-mass spectrometry, and principal component analysis as well as targeted analysis of known TRPA1 agonists. Activation of TRPA1 was attributable to both particle-associated electrophiles and non-electrophilic agonists, which affected the induction of interleukin-8 mRNA via TRPA1 in A549 and IMR-90 lung cells as well as TRPA1-mediated mucin gene induction in human lung cells and mucous cell metaplasia in mice. This work illustrates that not all DEP samples are equivalent, and studies aimed at assessing mechanisms of DEP toxicity should account for multiple variables, including the expression of receptor targets such as TRPA1 and particle chemistry.

OBJECTIVE: Redox-sensitive green fluorescent protein (roGFP) is a genetically-encoded redox-sensitive protein used to detect cellular oxidative stress associated with reactive oxygen species production. Here we replaced the cysteine at position 147 of roGFP1 (variant of roGFP) with selenocysteine in order to increase redox sensitivity of the redox reporter. RESULTS: Expression of roGFP1 selenoprotein (roGFP1-Se147) in HEK293 cells required the presence of a selenocysteine insertion sequence and was augmented by co-expression with SBP2. roGFP1-Se147 demonstrated a similar excitation and emission spectra to roGFP1. Although expression of roGFP1-Se147 was limited, it was sufficient enough to perform live cell imaging to evaluate sensitivity to oxidation and reduction. roGFP1-Se147 exhibited a 100-fold increase in sensitivity to oxidation with H2O2 in comparison to roGFP1 as well as a 20-fold decrease in the EC50 of H2O2. Furthermore, roGFP1-Se147, unlike roGFP1, was able to detect oxidation caused by the mitochondrial electron transport complex III inhibitor antimycin A. Unfortunately roGFP-Se147 exhibited a diminished dynamic range and photoinstability.

Inflammation causes nociceptive sensory neuron activation, evoking debilitating symptoms and reflexes. Inflammatory signaling pathways are capable of modulating mitochondrial function, resulting in reactive oxygen species (ROS) production, mitochondrial depolarization and calcium release. Previously we showed that mitochondrial modulation with antimycin A, a complex III inhibitor, selectively stimulated nociceptive bronchopulmonary C-fibers via the activation of transient receptor potential (TRP) ankyrin 1 (A1) and vanilloid 1 (V1) cation channels. TRPA1 is ROS-sensitive, but there is little evidence that TRPV1 is activated by ROS. Here, we used dual imaging of dissociated vagal neurons to investigate the correlation of mitochondrial superoxide production (mitoSOX) or mitochondrial depolarization (JC-1) with cytosolic calcium (Fura-2AM), following mitochondrial modulation by antimycin A, rotenone (complex I inhibitor) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP, mitochondrial uncoupling agent). Mitochondrial modulation by all agents selectively increased cytosolic calcium in a subset of TRPA1/TRPV1-expressing (A1/V1+) neurons. There was a significant correlation between antimycin A-induced calcium responses and mitochondrial superoxide in wild-type 'responding' A1/V1+ neurons, which was eliminated in TRPA1-/- neurons, but not TRPV1-/- neurons. Nevertheless, antimycin A-induced superoxide production did not always increase calcium in A1/V1+ neurons, suggesting a critical role of an unknown factor. CCCP caused both superoxide production and mitochondrial depolarization but neither correlated with calcium fluxes in A1/V1+ neurons. Rotenone-induced calcium responses in 'responding' A1/V1+ neurons correlated with mitochondrial depolarization but not superoxide production. Our data are consistent with the hypothesis that mitochondrial dysfunction causes calcium fluxes in a subset of A1/V1+ neurons via ROS-dependent and ROS-independent mechanisms.

Activation of the sensory nerve ion channel TRPA1 by electrophiles is the key mechanism that initiates nociceptive signaling, and leads to defensive reflexes and avoidance behaviors, during oxidative stress in mammals. TRPA1 is rapidly activated by subtoxic levels of electrophiles, but it is unclear how TRPA1 outcompetes cellular antioxidants that protect cytosolic proteins from electrophiles. Here, using physiologically relevant exposures, we demonstrate that electrophiles react with cysteine residues on mammalian TRPA1 at rates that exceed the reactivity of typical cysteines by 6,000-fold and that also exceed the reactivity of antioxidant enzymes. We show that TRPA1 possesses a complex reactive cysteine profile in which C621 is necessary for electrophile-induced binding and activation. Modeling of deprotonation energies suggests that K620 contributes to C621 reactivity and mutation of K620 alone greatly reduces the effect of electrophiles on TRPA1. Nevertheless, binding of electrophiles to C621 is not sufficient for activation, which also depends on the function of another reactive cysteine (C665). Together, our results demonstrate that TRPA1 acts as an effective electrophilic sensor because of the exceptionally high reactivity of C621.