Publications

The cough reflex is evoked by noxious stimuli in the airways. Although this reflex is essential for health, it can be triggered chronically in inflammatory and infectious airway disease. Neuronal transient receptor potential (TRP) channels such as ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) are polymodal receptors expressed on airway nociceptive afferent nerves. Reactive oxygen species (ROS) and other reactive compounds are associated with inflammation, from either NADPH oxidase or mitochondria. These reactive compounds cause activation and hyperexcitability of nociceptive afferents innervating the airways, and evidence suggests key contributions of TRPA1 and TRPV1.

Acute inhalation of airborne pollutants alters cardiovascular function and evidence suggests that pollutant-induced activation of airway sensory nerves via the gating of ion channels is critical to these systemic responses. Here, we have investigated the effect of capsaicin [transient receptor potential (TRP) vanilloid 1 (TRPV1) agonist], AITC [TRP ankyrin 1 (TRPA1) agonist], and ATP (P2X2/3 agonist) on bronchopulmonary sensory activity and cardiovascular responses of conscious Sprague-Dawley (SD) rats. Single fiber recordings show that allyl isothiocyanate (AITC) and capsaicin selectively activate C fibers, whereas subpopulations of both A and C fibers are activated by stimulation of P2X2/3 receptors. Inhalation of the agonists by conscious rats caused significant bradycardia, atrioventricular (AV) block, and prolonged PR intervals, although ATP-induced responses were lesser than those evoked by AITC or capsaicin. Responses to AITC were inhibited by the TRP channel blocker ruthenium red and the muscarinic antagonist atropine. AITC inhalation also caused a biphasic blood pressure response: a brief hypertensive phase followed by a hypotensive phase. Atropine accentuated the hypertensive phase, while preventing the hypotension. AITC-evoked bradycardia was not abolished by terazosin, the alpha1-adrenoceptor inhibitor, which prevented the hypertensive response. Anesthetics had profound effects on AITC-evoked bradycardia and AV block, which was abolished by urethane, ketamine, and isoflurane. Nevertheless, AITC inhalation caused bradycardia and AV block in paralyzed and ventilated rats following precollicular decerebration. In conclusion, we provide evidence that activation of ion channels expressed on nociceptive airway sensory nerves causes significant cardiovascular effects in conscious SD rats via reflex modulation of the autonomic nervous system.

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.

The Thy1.2 YFP-16 mouse expresses yellow fluorescent protein (YFP) in specific subsets of peripheral and central neurons. The original characterization of this model suggested that YFP was expressed in all sensory neurons, and this model has been subsequently used to study sensory nerve structure and function. Here, we have characterized the expression of YFP in the sensory ganglia (DRG, trigeminal and vagal) of the Thy1.2 YFP-16 mouse, using biochemical, functional and anatomical analyses. Despite previous reports, we found that YFP was only expressed in approximately half of DRG and trigeminal neurons and less than 10% of vagal neurons. YFP-expression was only found in medium and large-diameter neurons that expressed neurofilament but not TRPV1. YFP-expressing neurons failed to respond to selective agonists for TRPV1, P2X(2/3 and TRPM8 channels in Ca2+ imaging assays. Confocal analysis of glabrous skin, hairy skin of the back and ear and skeletal muscle indicated that YFP was expressed in some peripheral terminals with structures consistent with their presumed non-nociceptive nature. In summary, the Thy1.2 YFP-16 mouse expresses robust YFP expression in only a subset of sensory neurons. But this mouse model is not suitable for the study of nociceptive nerves or the function of such nerves in pain and neuropathies.

Excessive activation of the cough reflex is a major clinical problem in respiratory diseases. The cough reflex is triggered by activation of nociceptive sensory nerve terminals innervating the airways by noxious stimuli. Oxidative stress is a noxious stimuli associated with inhalation of pollutants and inflammatory airway disease. Here, we discuss recent findings that oxidative stress, in particular downstream of mitochondrial dysfunction, evokes increased electrical activity in airway nociceptive sensory nerves. Mechanisms include activation of transient receptor potential (TRP) channels and protein kinase C. Such mechanisms may contribute to excessive cough reflexes in respiratory diseases.

Cough is a reflex that serves to protect the airways. Excessive or chronic coughing is a major health issue that is poorly controlled by current therapeutics. Significant effort has been made to understand the mechanisms underlying the cough reflex. The focus of this review is the evidence supporting the role of specific airway sensory nerve (afferent) populations in the initiation and modulation of the cough reflex in health and disease.

Persons with allergies present with symptoms that often are the result of alterations in the nervous system. Neuronally based symptoms depend on the organ in which the allergic reaction occurs but can include red itchy eyes, sneezing, nasal congestion, rhinorrhea, coughing, bronchoconstriction, airway mucus secretion, dysphagia, altered gastrointestinal motility, and itchy swollen skin. These symptoms occur because mediators released during an allergic reaction can interact with sensory nerves, change processing in the central nervous system, and alter transmission in sympathetic, parasympathetic, and enteric autonomic nerves. In addition, evidence supports the idea that in some subjects this neuromodulation is, for reasons poorly understood, upregulated such that the same degree of nerve stimulus causes a larger effect than seen in healthy subjects. There are distinctions in the mechanisms and nerve types involved in allergen-induced neuromodulation among different organ systems, but general principles have emerged. The products of activated mast cells, other inflammatory cells, and resident cells can overtly stimulate nerve endings, cause long-lasting changes in neuronal excitability, increase synaptic efficacy, and also change gene expression in nerves, resulting in phenotypically altered neurons. A better understanding of these processes might lead to novel therapeutic strategies aimed at limiting the suffering of those with allergies.

Airway sensory nerve excitability is a key determinant of respiratory disease-associated reflexes and sensations such as cough and dyspnea. Inflammatory signaling modulates mitochondrial function and produces reactive oxygen species (ROS). Peripheral terminals of sensory nerves are densely packed with mitochondria; thus, we hypothesized that mitochondrial modulation would alter neuronal excitability. We recorded action potential firing from the terminals of individual bronchopulmonary C-fibers using a mouse ex vivo lung-vagal ganglia preparation. C-fibers were characterized as nociceptors or non-nociceptors based upon conduction velocity and response to transient receptor potential (TRP) channel agonists. Antimycin A (mitochondrial complex III Qi site inhibitor) had no effect on the excitability of non-nociceptors. However, antimycin A increased excitability in nociceptive C-fibers, decreasing the mechanical threshold by 50% and increasing the action potential firing elicited by a P2X2/3 agonist to 270% of control. Antimycin A-induced nociceptor hyperexcitability was independent of TRP ankyrin 1 or TRP vanilloid 1 channels. Blocking mitochondrial ATP production with oligomycin or myxothiazol had no effect on excitability. Antimycin A-induced hyperexcitability was dependent on mitochondrial ROS and was blocked by intracellular antioxidants. ROS are known to activate protein kinase C (PKC). Antimycin A-induced hyperexcitability was inhibited by the PKC inhibitor bisindolylmaleimide (BIM) I, but not by its inactive analog BIM V. In dissociated vagal neurons, antimycin A caused ROS-dependent PKC translocation to the membrane. Finally, H2O2 also induced PKC-dependent nociceptive C-fiber hyperexcitability and PKC translocation. In conclusion, ROS evoked by mitochondrial dysfunction caused nociceptor hyperexcitability via the translocation and activation of PKC.

Mitochondrial dysfunction and subsequent oxidative stress has been reported for a variety of cell types in inflammatory diseases. Given the abundance of mitochondria at the peripheral terminals of sensory nerves and the sensitivity of transient receptor potential (TRP) ankyrin 1 (A1) and TRP vanilloid 1 (V1) to reactive oxygen species (ROS) and their downstream products of lipid peroxidation, we investigated the effect of nerve terminal mitochondrial dysfunction on airway sensory nerve excitability. Here we show that mitochondrial dysfunction evoked by acute treatment with antimycin A (mitochondrial complex III Qi site inhibitor) preferentially activated TRPA1-expressing "nociceptor-like" mouse bronchopulmonary C-fibers. Action potential discharge was reduced by the TRPA1 antagonist HC-030031. Inhibition of TRPV1 further reduced C-fiber activation. In mouse dissociated vagal neurons, antimycin A induced Ca(2+) influx that was significantly reduced by pharmacological inhibition or genetic knockout of either TRPA1 or TRPV1. Inhibition of both TRPA1 and TRPV1 was required to abolish antimycin A-induced Ca(2+) influx in vagal neurons. Using an HEK293 cell expression system, antimycin A induced concentration-dependent activation of both hTRPA1 and hTRPV1 but failed to activate nontransfected cells. Myxothiazol (complex III Qo site inhibitor) inhibited antimycin A-induced TRPA1 activation, as did the reducing agent dithiothreitol. Scavenging of both superoxide and hydrogen peroxide inhibited TRPA1 activation following mitochondrial modulation. In conclusion, we present evidence that acute mitochondrial dysfunction activates airway sensory nerves preferentially via TRPA1 through the actions of mitochondrially-derived ROS. This represents a novel mechanism by which inflammation may be transduced into nociceptive electrical signaling.

Vagal sensory nerves innervate the majority of visceral organs (e.g., heart, lungs, GI tract, etc) and their activation is critical for defensive and regulatory reflexes. Intracellular Ca(2+) is a key regulator of neuronal excitability and is largely controlled by the Ca(2+) stores of the endoplasmic reticulum. In other cell types store-operated channels (SOC) have been shown to contribute to the homeostatic control of intracellular Ca(2+). Here, using Ca(2+) imaging, we have shown that ER depletion in vagal sensory neurons (using thapsigargin or caffeine) in the absence of extracellular Ca(2+) evoked Ca(2+) influx upon re-introduction of Ca(2+) into the extracellular buffer. This store-operated Ca(2+) entry (SOCE) was observed in approximately 25-40% of vagal neurons, equally distributed among nociceptive and non-nociceptive sensory subtypes. SOCE was blocked by Gd(3+) but not by the Orai channel blocker SKF96365. We found Orai channel mRNA in extracts from whole vagal ganglia, but when using single cell RT-PCR analysis we found only 3 out of 34 neurons expressed Orai channel mRNA, indicating that Orai channel expression in the vagal ganglia was likely derived from non-neuronal cell types. Confocal microscopy of vagal neurons in 3 day cultures demonstrated rich ER tracker fluorescence throughout axonal and neurite structures and ER store depletion (thapsigargin) evoked Ca(2+) transients from these structures. However, no SOCE could be detected in the axonal/neurite structures of vagal neurons. We conclude that SOCE occurs in vagal sensory neuronal cell bodies through non-Orai mechanisms but is absent at nerve terminals.