Pulmonary Sensory Neurons Research Articles

Evidence for vagal sensory neural involvement in influenza pathogenesis and disease.

Influenza A virus (IAV) is a common respiratory pathogen and a global cause of significant and often severe morbidity. Although inflammatory immune responses to IAV infections are well described, little is known about how neuroimmune processes contribute to IAV pathogenesis. In the present study, we employed surgical, genetic, and pharmacological approaches to manipulate pulmonary vagal sensory neuron innervation and activity in the lungs to explore potential crosstalk between pulmonary sensory neurons and immune processes. Intranasal inoculation of mice with H1N1 strains of IAV resulted in stereotypical antiviral lung inflammation and tissue pathology, changes in breathing, loss of body weight and other clinical signs of severe IAV disease. Unilateral cervical vagotomy and genetic ablation of pulmonary vagal sensory neurons had a moderate effect on the pulmonary inflammation induced by IAV infection, but significantly worsened clinical disease presentation. Inhibition of pulmonary vagal sensory neuron activity via inhalation of the charged sodium channel blocker, QX-314, resulted in a moderate decrease in lung pathology, but again this was accompanied by a paradoxical worsening of clinical signs. Notably, vagal sensory ganglia neuroinflammation was induced by IAV infection and this was significantly potentiated by QX-314 administration. This vagal ganglia hyperinflammation was characterized by alterations in IAV-induced host defense gene expression, increased neuropeptide gene and protein expression, and an increase in the number of inflammatory cells present within the ganglia. These data suggest that pulmonary vagal sensory neurons play a role in the regulation of the inflammatory process during IAV infection and suggest that vagal neuroinflammation may be an important contributor to IAV pathogenesis and clinical presentation. Targeting these pathways could offer therapeutic opportunities to treat IAV-induced morbidity and mortality.

Mesenchymal stromal cells alleviate depressive and anxiety-like behaviors via a lung vagal-to-brain axis in male mice

Major depressive disorder (MDD) is one of the most common and disabling mental disorders, and current strategies remain inadequate. Although mesenchymal stromal cells (MSCs) have shown beneficial effects in experimental models of depression, underlying mechanisms remain elusive. Here, using murine depression models, we demonstrated that MSCs could alleviate depressive and anxiety-like behaviors not due to a reduction in proinflammatory cytokines, but rather activation of dorsal raphe nucleus (DRN) 5-hydroxytryptamine (5-HT) neurons. Mechanistically, peripheral delivery of MSCs activated pulmonary innervating vagal sensory neurons, which projected to the nucleus tractus solitarius, inducing the release of 5-HT in DRN. Furthermore, MSC-secreted brain-derived neurotrophic factor activated lung sensory neurons through tropomyosin receptor kinase B (TrkB), and inhalation of a TrkB agonist also achieved significant therapeutic effects in male mice. This study reveals a role of peripheral MSCs in regulating central nervous system function and demonstrates a potential “lung vagal-to-brain axis” strategy for MDD.

Rapid Intravenous Injection of Fentanyl (FNT) Causes a Vagal‐Mediated Lethal Sudden Ventilatory Arrest in Anesthetized Rats

Opioids have been used for analgesia and anesthesia mainly via activation of central opioid mu‐receptors (MORs). It also causes overdose deaths due to ventilatory failure, in which FNT is the deadest. Rapid intravenous (IV) injection of overdose FNT triggers a sudden death within a few minutes or even seconds, but the relevant mechanism remains unclear. Because bolus IV injection of FNT at a low dose produced a vagal‐mediated brief apnea (Zhuang et al, AJP 2012), This study was aimed to characterize the cardiorespiratory disorders responsible for the sudden death and determine the vagal role in generating the lethal ventilatory arrest. EMGs of external and internal intercostal (EMGEI and EMGII), thyroarytenoid and superior pharyngeal constrictor muscles (EMGTA and EMGSPC) were simultaneously recorded before and after IV injection of FNT in the anesthetized vagal intact and vagotomized rats. The optical fiber scope of an oral video camera was applied to visualize the vocal and pharyngeal responses. Immunohistochemical approach was used to define the presence of MOR expression in the pulmonary vagal sensory neurons retrogradely labeled by DiI. FNT induced an apnea or a lethal ventilatory arrest in a dose‐dependent manner associated with hypotension and bradycardia. The apnea/ventilatory arrest was characterized by silence of EMGEI with tonic discharges of EMGII, EMGTA, and EMGSPC, i.e., the central apnea (expiratory) coupled with vocal closure and pharyngeal constriction/collapse and chest wall rigidity (triple‐apnea). Vagotomy abolished the evoked cardiorespiratory responses. MOR expression in vagal pulmonary neurons, especially C‐neurons of the nodose and jugular ganglia. Our results suggest that rapid IV injection of FNT at a high dose uniquely causes a vagal‐mediated triple‐apnea, leading to the sudden death in anesthetized rats.

Mechanisms Underlying the Stimulatory Effect of Inhaled Sulfur Dioxide on Rat Vagal Bronchopulmonary Sensory Nerves

Chronic exposure to sulfur dioxide (SO2), an air pollutant, can cause airway injury and lung diseases. Acute exposure to SO2 triggers cough and reflex bronchoconstriction, indicating a stimulatory effect of SO2 on airway sensory nerves. Indeed, a recent study in our lab has demonstrated that vagal bronchopulmonary C‐fibers are the primary target of inhaled SO2. This study was carried out to investigate the underlying mechanism of this stimulatory effect of SO2 on bronchopulmonary C‐fibers. Our results showed: 1) Inhalation of SO2 (1,000–2,000 ppm, 10 breaths) evoked a pronounced and reproducible stimulatory effect on pulmonary C‐fibers in anesthetized rats. 2) Pretreatment with an intravenous infusion of sodium bicarbonate (120 μmol/kg) raised the baseline arterial pH by 0.08 units, which profoundly diminished the stimulatory effect of SO2 on pulmonary C‐fibers by 83.9%. 3) This stimulatory effect of SO2 was also significantly attenuated by a pretreatment with amiloride (a blocker of acid‐sensing ion channels, ASICs; 11 μmol/kg) alone (Δ = 70.7%); AMG9810 (an antagonist of transient receptor potential vanilloid type‐1, TRPV1; 10 μmol/kg) alone (Δ = 76.7%); and by a combination of amiloride and AMG9810 (Δ = 91.4%). 4) To further investigate if this stimulatory effect is generated by a direct action of SO2 on sensory nerves, the change in intracellular Ca2+ concentration, [Ca2+]i, was measured in isolated rat vagal pulmonary sensory neurons. Perfusion with extracellular fluid saturated with SO2 (1,000 and 2,000 ppm) evoked a significant increase in [Ca2+]i in a concentration‐dependent manner in these neurons, and these responses to SO2 were markedly suppressed by a combined pretreatment with amiloride and AMG9810. 5) In addition, inhalation of SO2 (300 and 600 ppm for 5 min) evoked cough reflex responses consistently in awake mice in a concentration‐dependent manner; the cough responses were almost completely prevented by a pretreatment with amiloride aerosol inhalation (5 mM for 4 min) in TRPV1‐knockout mice. In conclusion, this study suggested that inhalation of SO2 lowered the pH in airway/lung tissues, which exerted a stimulatory effect on vagal bronchopulmonary C‐fibers by activating both ASICs and TRPV1 channels.Support or Funding InformationNIH grants AI123832 & UL1TR001998This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TRPA1‐Induced Pulmonary Spinal Sympathetic Afferent Activation is Attenuated in Rats with Chronic Heart Failure

In a recent preliminary study (Association of the University Anesthesiologists 64th annual meeting, Abstract #1109, 2017), we reported that activation of pulmonary spinal afferents by topical application of bradykinin (BK) and the TRPV1 agonist capsaisin on to lung surfaces (especially on the ventral surface) evokes a previously undocumented potent sympatho‐excitatory reflex that includes increased bood pressure (BP), heart rate (HR) and renal sympathetic nerve activity (RSNA) in urethane‐α‐chloralose anesthetized, vagotomized rats. These data suggested a new pulmonary sympathetic afferent reflex (PSAR) in rats. However, it remains unclear whether pulmonary sympathetic afferents can also be activated by the transient receptor potential ankyrin 1 (TRPA1) agonist, allyl isothiocyanate (AITC). In the current study, we examined the TRPA1‐induced PSAR in both normal and chronic heart failure (CHF) states. After bilateral vagotomy, we applied filter paper (3 × 3 mm) saturated with 30 mM AITC to the ventral lung surfaces to stimulate the PSAR in urethane‐ α‐chloralose anesthetized sham‐operated and CHF (8 weeks post myocardial infarction) rats. We found that application of AITC to the lungs resulted in a biphasic BP response whereas HR and RSNA remained elevated during the AITC application in sham rats (Table 1). Compared to sham rats, AITC caused a predominantly depressor response in CHF rats. The increases in HR and RSNA in response to AITC were also attenuated in CHF rats. These data suggest that activation of the TRPA1 receptor in pulmonary spinal afferents can cause sympatho‐excitation in normal rats whereas this response was blunted in CHF rats. Ongoing studies are validating TRPA1 receptor protein expression in pulmonary sensory neurons in sham and CHF rats.Support or Funding InformationThis work was, in part, supported by grants from the National Heart, Lung, and Blood Institute (1R01HL121012‐01 and 1R01HL126796‐01).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exposure to Leukotriene E4 Induces Hypersensitivity of Vagal Pulmonary Sensory Neurons in Rats

Leukotrienes E4 (LTE4), the most stable and abundant cysteinyl leukotrienes (CysLTs), has been generally studied less than other two CysLTs (LTC4 and LTD4), likely because it is only a weak agonist for the classical type 1 (CysLT1) and type 2 (CysLT2) receptors for CysLTs. However, recent identification of GRP99 protein as a high affinity receptor for LTE4 suggests that LTE4 may play a more pivotal role in the pathogenesis of airway inflammatory diseases such as asthma. The present study is carried out to investigate the effect of LTE4 on the chemo‐sensitivity of pulmonary afferent neurons in both whole‐animal ventilatory and single neuron electrophysiological recordings. In anesthetized spontaneously breathing rats, intratracheal instillation of LTE4 (2 nmol in 100 μl saline) significantly potentiates chemoreflexes evoked by right‐atrial injection of capsaicin, a specific agonist for transient receptor potential vanilloid receptor 1 (TRPV1). In patch‐clamp recordings with isolated rat vagal pulmonary sensory neurons, pretreatment with LTE4 concentration‐dependently enhances capsaicin‐induced TRPV1‐mediated whole‐cell inward currents. Our patch‐clamp experiments further demonstrate that the zinc‐evoked TRPA1‐mediated current is also markedly enhanced in these neurons. In summary, our data show that exposure to LTE4 leads to hypersensitivity of vagal pulmonary sensory neurons and augments pulmonary chemoreflexes in rats.Support or Funding InformationSupported by MUSM and Navicent Health FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Toll-like receptor expression in pulmonary sensory neurons in the bleomycin-induced fibrosis model.

Airway sensory nerves are known to express several receptors and channels that are activated by exogenous and endogenous mediators that cause coughing. Toll-like receptor (TLR) s are expressed in nociceptive neurons and play an important role in neuroinflammation. However, there have been very few studies of TLR expression in lung-derived sensory neurons or their relevance to respiratory symptoms such as cough. We used the bleomycin-induced pulmonary fibrosis model to investigate the change in TLR expression in pulmonary neurons and the association of TLRs with transient receptor potential (TRP) channels in pulmonary neurons. After 2 weeks of bleomycin or saline administration, pulmonary fibrosis changes were confirmed using tissue staining and the SIRCOL collagen assay. TLRs (TLR 1–9) and TRP channel expression was analyzed using single cell reverse transcription polymerase chain reaction (RT-PCR) in isolated sensory neurons from the nodose/jugular ganglion and the dorsal root ganglion (DRG). Pulmonary sensory neurons expressed TLR2 and TLR5. In the bleomycin-induced pulmonary fibrosis model, TLR2 expression was detected in 29.5% (18/61) and 26.9% (21/78) of pulmonary nodose/jugular neurons and DRG neurons, respectively. TLR5 was also detected in 55.7% (34/61) and 42.3% (33/78) of pulmonary nodose/jugular neurons and DRG neurons, respectively, in the bleomycin-induced pulmonary fibrosis model. TLR5 was expressed in 63.4% of TRPV1 positive cells and 43.4% of TRPM8 positive cells. In conclusion, TLR2 and TLR5 expression is enhanced, especially in vagal neurons, in the bleomycin-induced fibrosis model group when compared to the saline treated control group. Co-expression of TLR5 and TRP channels in pulmonary sensory neurons was also observed. This work sheds new light on the role of TLRs in the control and manifestation of clinical symptoms, such as cough. To understand the role of TLRs in pulmonary sensory nerves, further study will be required.

Hypersensitivity of Vagal Pulmonary Afferents Induced by Tumor Necrosis Factor Alpha in Mice.

Tumor necrosis factor alpha (TNFα), a pro-inflammatory cytokine, plays a significant role in the pathogenesis of allergic asthma. Inhalation of TNFα also induces airway hyperresponsiveness in healthy human subjects, and the underlying mechanism is not fully understood. A recent study reported that TNFα caused airway inflammation and a sustained elevation of pulmonary chemoreflex responses in mice, suggesting a possible involvement of heightened sensitivity of vagal pulmonary C-fibers. To investigate this possibility, the present study aimed to investigate the effect of a pretreatment with TNFα on the sensitivity of vagal pulmonary afferents in anesthetized mice. After TNFα (10 μg/ml, 0.03 ml) and vehicle (Veh; phosphate buffered saline (PBS), 0.03 ml) were administered by intra-tracheal instillation in each mouse of treated (TNF) and control (Veh) groups, respectively, the peak activity of pulmonary C-fibers in response to an intravenous bolus injection of a low dose of capsaicin (Cap; 0.5 μg/kg) was significantly elevated in TNF group (6.5 ± 1.3 impulses/s, n = 12) 24–48 h later, compared to that in Veh group (2.2 ± 0.5 impulses/s, n = 11; P < 0.05). Interestingly, the same low dose of Cap injection also evoked a distinct burst of discharge (2.4 ± 0.7 impulses/s) in 75% of the silent rapidly adapting receptors (RARs), a subtype of RARs exhibiting no phasic activity, in TNF group, but did not stimulate any of the silent RARs in Veh group. To further determine if this sensitizing effect involves a direct action of TNFα on these sensory nerves, the change in intracellular Ca2+ concentration in response to Cap challenge was measured in isolated mouse vagal pulmonary sensory neurons. The Cap-evoked Ca2+ influx was markedly enhanced in the neurons incubated with TNFα (50 ng/ml) for ~24 h, and this sensitizing effect was attenuated in the neurons isolated from the TNF-receptor double homozygous mutant mice. In conclusion, the TNFα pretreatment enhanced the Cap sensitivity in both pulmonary C-fibers and silent RARs, and the action was mediated through TNF receptors. These sensitizing effects of TNFα may contribute, at least in part, to the pathogenesis of airway hyperresponsiveness induced by this cytokine.

Immediate and delayed potentiating effects of tumor necrosis factor-α on TRPV1 sensitivity of rat vagal pulmonary sensory neurons.

We studied acute effects of tumor necrosis factor-α (TNFα) on the sensitivity of isolated rat vagal pulmonary sensory neurons. Our results showed the following. First, a brief pretreatment with a low dose of TNFα (1.44 nM, 9 min) enhanced the sensitivity of transient receptor potential vanilloid type 1 (TRPV1) receptors in these neurons in two distinct phases: the inward current evoked by capsaicin was amplified (Δ = 247%) immediately following the TNFα pretreatment, which gradually declined toward control and then increased again reaching another peak (Δ = 384%) after 60-90 min. Second, the immediate phase of this potentiating effect of TNFα was completely abolished by a pretreatment with a selective cyclooxygenase-2 (COX-2) inhibitor, NS-398, whereas the delayed potentiation was only partially attenuated. Third, in sharp contrast, TNFα did not generate any potentiating effect on the responses to non-TRPV1 chemical activators of these neurons. Fourth, the selectivity of the TNFα action on TRPV1 was further illustrated by the responses to acid (pH 6.0); TNFα did not affect the rapid transient current mediated by acid-sensing ion channels but significantly augmented the slow sustained current mediated by TRPV1 in the same neurons. Fifth, in anesthetized rats, a similar pattern of acute sensitizing effects of TNFα on pulmonary C-fiber afferents and the involvement of COX-2 were also clearly shown. In conclusion, a brief pretreatment with TNFα induced both immediate and delayed potentiating effects on the TRPV1 sensitivity in pulmonary sensory neurons, and the production of COX-2 arachidonic acid metabolites plays a major role in the immediate sensitizing effect of TNFα.

Activation of bitter taste receptors in pulmonary nociceptors sensitizes TRPV1 channels through the PLC and PKC signaling pathway.

Bitter taste receptors (T2Rs), a G protein-coupled receptor family capable of detecting numerous bitter-tasting compounds, have recently been shown to be expressed and play diverse roles in many extraoral tissues. Here we report the functional expression of T2Rs in rat pulmonary sensory neurons. In anesthetized spontaneously breathing rats, intratracheal instillation of T2R agonist chloroquine (10 mM, 0.1 ml) significantly augmented chemoreflexes evoked by right-atrial injection of capsaicin, a specific activator for transient receptor potential vanilloid receptor 1 (TRPV1), whereas intravenous infusion of chloroquine failed to significantly affect capsaicin-evoked reflexes. In patch-clamp recordings with isolated rat vagal pulmonary sensory neurons, pretreatment with chloroquine (1-1,000 µM, 90 s) concentration dependently potentiated capsaicin-induced TRPV1-mediated inward currents. Preincubating with diphenitol and denatonium (1 mM, 90 s), two other T2R activators, also enhanced capsaicin currents in these neurons but to a lesser extent. The sensitizing effect of chloroquine was effectively prevented by the phospholipase C inhibitor U73122 (1 µM) or by the protein kinase C inhibitor chelerythrine (10 µM). In summary, our study showed that activation of T2Rs augments capsaicin-evoked TRPV1 responses in rat pulmonary nociceptors through the phospholipase C and protein kinase C signaling pathway.

Molecular Diversity of Pulmonary Sensory Neurons Revealed by Single Cell RNA Sequencing

Pulmonary sensory neurons are primary sensory neurons that monitor lung physiological state, and communicate that information to the central nervous system. Many symptoms associated with pulmonary diseases, such as chronic cough, dyspnea (breathlessness), and airway hyperresponsiveness, are presumed to result from abnormal behaviors of these neurons. Past studies indicate that pulmonary sensory neurons are heterogeneous in their morphologies, functions and molecular identities. However, a complete understanding of their molecular and functional diversity is still lacking. Here, we perform single cell RNA sequencing (scRNA‐seq) on mouse pulmonary sensory neurons to elucidate the genome‐wide expression profile of over a hundred individual pulmonary sensory neurons. Through unbiased clustering analysis of the expression data of these neurons, we define 7 major classes of pulmonary sensory neurons. Each class expresses distinct sets of genes implicated in physiological sensing, which predict the specific sensory functions of each class. The gene expression profiles also identify specific molecular markers for each class, which we are using to elucidate their structures, map their terminal locations and receptive fields in the lung, measure their activity, identify their synaptic partners, and reveal the physiological consequences of their activation.Support or Funding InformationDamon Runyon Postdoctoral Research Fellowship (Y.L.)M.A.K. is an investigator of the Howard Hughes Medical Institute

Activation of Bitter Taste Receptors in Rat Pulmonary Sensory Neurons Augments Capsaicin‐Evoked TRPV1 Responses

Bitter taste receptors (T2Rs), a G‐protein‐coupled receptor family capable of detecting hundreds of bitter‐tasting compounds, have recently been shown to be expressed and play diverse roles in various extraoral tissues. Here we report the functional expression of T2Rs in rat pulmonary sensory neurons. In anesthetized spontaneously breathing rats, intratracheal instillation of T2Rs agonist chloroquine (10 mM, 0.1 ml) significantly augmented the chemoreflex responses evoked by right‐atrial injection of capsaicin, a specific activator for transient receptor potential vanilloid receptor 1 (TRPV1); whereas intravenous infusion of chloroquine failed to significantly affect capsaicin‐evoked reflexes. In patch‐clamp recordings with isolated rat vagal pulmonary sensory neurons, pretreatment with chloroquine (1–1000 μM, 90 s) concentration‐dependently potentiated capsaicin‐induced inward currents. This sensitizing effect of chloroquine was effectively blocked by phospholipase C inhibitor U73122 (0.75 μM), or by protein kinase C inhibitor chelerythrine (5 μM). In summary, our study showed that activation of T2Rs augments capsaicin‐evoked TRPV1 responses in rat pulmonary sensory neurons, through phospholipase C and protein kinase C signaling pathway.Support or Funding InformationSupported by funds from MEDCEN and MUSM.

Interaction between TRPA1 and TRPV1: Synergy on pulmonary sensory nerves

Transient receptor potential ankyrin type 1 (TRPA1) and vanilloid type 1 (TRPV1) receptors are co-expressed in vagal pulmonary C-fiber sensory nerves. Because both these ligand-gated non-selective cation channels are sensitive to a number of endogenous inflammatory mediators, it is highly probable that they can be activated simultaneously during airway inflammation. Studies were carried out to investigate whether there is an interaction between these two polymodal transducers upon simultaneous activation, and how it modulates the activity of vagal pulmonary C-fiber sensory nerves. Our studies showed a distinct potentiating effect induced abruptly by simultaneous activations of TRPA1 and TRPV1 by their respective selective agonists, allyl isothiocyanate (AITC) and capsaicin (Cap), at near-threshold concentrations. This synergistic effect was demonstrated in the studies of single-unit recording of vagal bronchopulmonary C-fiber afferents and the reflex responses elicited by activation of these afferents in intact animals, as well as in the isolated nodose and jugular bronchopulmonary sensory neurons. This potentiating effect was absent when either AITC or Cap was replaced by non-TRPA1 and non-TRPV1 chemical activators of these neurons, demonstrating the selectivity of the interaction between these two TRP channels. Furthermore, the synergism was dependent upon the extracellular Ca2+, and the rapid onset of the action further suggests that the interaction probably occurred locally at the sites of these channels. These findings suggest that the TRPA1–TRPV1 interaction may play an important role in regulating the function and excitability of pulmonary sensory neurons during airway inflammation, but the mechanism underlying this positive interaction is not yet fully understood.

Role of calcium ions in the positive interaction between TRPA1 and TRPV1 channels in bronchopulmonary sensory neurons.

Both transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors are abundantly expressed in bronchopulmonary C-fiber sensory nerves and can be activated by a number of endogenous inflammatory mediators. A recent study has reported a synergistic effect of simultaneous TRPA1 and TRPV1 activations in vagal pulmonary C-fiber afferents in anesthetized rats, but its underlying mechanism was not known. This study aimed to characterize a possible interaction between these two TRP channels and to investigate the potential role of Ca(2+) as a mediator of this interaction in isolated rat vagal pulmonary sensory neurons. Using the perforated patch-clamp recording technique, our study demonstrated a distinct positive interaction occurring abruptly between TRPA1 and TRPV1 when they were activated simultaneously by their respective agonists, capsaicin (Cap) and allyl isothiocyanate (AITC), at near-threshold concentrations in these neurons. AITC at this low concentration evoked only minimal or undetectable responses, but it markedly amplified the Cap-evoked current in the same neurons. This potentiating effect was eliminated when either AITC or Cap was replaced by non-TRPA1 and non-TRPV1 chemical activators of these neurons, demonstrating the selectivity of the interaction between these two TRP channels. Furthermore, when Ca(2+) was removed from the extracellular solution, the synergistic effect of Cap and AITC on pulmonary sensory neurons was completely abrogated, clearly indicating a critical role of Ca(2+) in mediating the action. These results suggest that this TRPA1-TRPV1 interaction may play a part in regulating the sensitivity of pulmonary sensory neurons during airway inflammatory reaction.

Positive Interaction between TRPA1 and TRPV1 Channels in Rat Vagal Bronchopulmonary Sensory Neurons

Both TRPA1 and TRPV1 are selectively and abundantly expressed in vagal bronchopulmonary C-fiber sensory nerves. They can be either activated or sensitized by a number of endogenous inflammatory mediators such as H+, ROS, bradykinin, protanoids, etc. As such, it is highly probable that concurrent activation of these two receptors can occur during airway inflammatory reaction. A recent study has demonstrated a distinct synergistic effect of simultaneous activation of TRPA1 and TRPV1 in vagal pulmonary C-fiber afferents in anesthetized rats (Lin et al., JAP, 2014), but its underlying mechanism and whether this action occurred directly at the sensory neurons were not known. This study was carried out to answer these questions in isolated nodose and jugular pulmonary sensory neurons. Using the perforated patch-clamp recording technique, our results showed: 1) The current density evoked by a combination of low concentrations of allyl isothiocyanate (AITC, a TRPA1 activator; 30 µM) and capsaicin (Cap, a TRPV1 activator; 0.1-0.3 µM) was 233% of the mathematical sum of the responses to AITC and Cap when they were administered separately at the same concentration and duration (4 sec) in the same neurons. 2) The positive interaction was absent when either AITC or Cap was replaced by another chemical activator of these neurons (e.g., ATP or phenylbiguanide). 3) This synergistic effect was completely blocked when Ca2+ was removed from the extracellular solution. 4) Application of AITC alone immediately preceding Cap also significantly augmented the Cap-evoked current, whereas reversing the order did not potentiate the response to AITC. In conclusion, a positive interaction occurs directly in vagal pulmonary sensory neurons between TRPA1 and TRPV1 when both are activated, and the extracellular Ca2+ plays an important role in this action.

A synergistic effect of simultaneous TRPA1 and TRPV1 activations on vagal pulmonary C-fiber afferents.

Transient receptor potential ankyrin type 1 (TRPA1) and vanilloid type 1 (TRPV1) receptors are coexpressed in vagal pulmonary C-fiber sensory nerves. Because both these receptors are sensitive to a number of endogenous inflammatory mediators, it is conceivable that they can be activated simultaneously during airway inflammation. This study aimed to determine whether there is an interaction between these two polymodal transducers upon simultaneous activation, and how it modulates the activity of vagal pulmonary C-fiber sensory nerves. In anesthetized, spontaneously breathing rats, the reflex-mediated apneic response to intravenous injection of a combined dose of allyl isothiocyanate (AITC, a TRPA1 activator) and capsaicin (Cap, a TRPV1 activator) was ∼202% greater than the mathematical sum of the responses to AITC and Cap when they were administered individually. Similar results were also observed in anesthetized mice. In addition, the synergistic effect was clearly demonstrated when the afferent activity of single vagal pulmonary C-fiber afferents were recorded in anesthetized, artificially ventilated rats; C-fiber responses to AITC, Cap and AITC + Cap (in combination) were 0.6 ± 0.1, 0.8 ± 0.1, and 4.8 ± 0.6 impulses/s (n = 24), respectively. This synergism was absent when either AITC or Cap was replaced by other chemical activators of pulmonary C-fiber afferents. The pronounced potentiating effect was further demonstrated in isolated vagal pulmonary sensory neurons using the Ca(2+) imaging technique. In summary, this study showed a distinct positive interaction between TRPA1 and TRPV1 when they were activated simultaneously in pulmonary C-fiber sensory nerves.

Acute potentiating effects of tumor necrosis factor‐α (TNFα) on the responses of rat vagal pulmonary sensory neurons to capsaicin challenge

TNFα is a proinflammatory cytokine, and plays an important role in the pathogenesis of allergic asthma. A recent study in our lab has demonstrated that a prolonged (24–48 h) treatment with TNFα increased the Ca2+ influx evoked by TRPV1 activators in vagal pulmonary sensory neurons (Am J Physiol 299: 483–492, 2010). The present study was carried out to test the acute effect of transient TNFα treatment on the TRPV1 sensitivity in isolated rat pulmonary sensory neurons. Our results showed: 1) A brief treatment with a low concentration of TNFα (1.44 nM; 9 min) induced a biphasic TRPV1 hypersensitivity in these neurons: the inward current evoked by capsaicin (average concentration 100 nM; 3 s) was enhanced both immediately following the TNFα treatment (Δ=280%), and after a long delay (peak at ~90 min; Δ=382%). 2) The immediate phase of the potentiating effect of TNFα was almost completely abolished by pretreatment with a COX‐2 inhibitor, NS‐398 (10 μM; 10 min), whereas the delayed phase was only partially attenuated. 3) In contrast, the same TNFα treatment did not generate any potentiating effect on the response to ATP (0.3 – 1.0 μM) challenge in these neurons. In summary, a brief treatment with TNFα induced both immediate and delayed potentiating effects on the TRPV1 sensitivity in isolated pulmonary sensory neurons, and a production of COX‐2 arachidonic acid metabolites is probably involved in the immediate sensitizing effect of TNFα. (Supported in part by NIH grant HL96914 &amp; NSC98‐2917‐I038‐101 fellowship from ROC)

Functional expression of calcium sensing receptor in rat vagal pulmonary sensory neurons

The calcium sensing receptor (CaSR), first isolated from bovine and human parathyroid, is a G‐protein‐coupled receptor that monitors the systemic, extracellular, free ionized‐calcium level in organs involved in systemic Ca2+ homeostasis. Recent studies have shown that CaSR is also expressed in many tissues that are not directly related to the plasma Ca2+ regulation, such as central and peripheral nervous systems where the function of this receptor remains to be defined. In this study, we examined the functional expression of CaSR in rat vagal pulmonary sensory neurons. Our immunohistochemical experiments demonstrated the expression of CaSR in these sensory neurons, as well as in the trachea and lungs. Whole‐cell patch‐clamp recordings were performed in isolated pulmonary sensory neurons. The results showed that pretreatment with NPS 2390 or NPS 2143, a selective CaSR antagonist, significantly enhanced the capsaicin‐evoked whole‐cell inward current. These data suggest that CaSR is likely involved in the integration of primary pulmonary sensory inputs under physiologic and/or pathophysiologic conditions. Funded partially by AHA 0835320N and MUSM startup funds.

House dust mite potentiates capsaicin‐evoked Ca2+ transients in mouse pulmonary sensory neurons via activation of protease‐activated receptor‐2

House dust mite (HDM) is a major source of allergen in house dust and has been suggested to be involved in the pathogenesis of asthma. In this study, we aimed to investigate whether HDM can modulate the sensitivity of pulmonary sensory neurons and, if so, to elucidate the underlying mechanism. Fura-2-based ratiometric Ca(2+) imaging was carried out to determine the effect of HDM extract on the capsaicin-evoked Ca(2+) transient in mouse vagal pulmonary sensory neurons. Pretreatment with HDM (50 μg ml(-1), 5 min) significantly enhanced the Ca(2+) transient evoked by capsaicin in these neurons isolated from wild-type mice. This potentiating effect of HDM was not antagonized by E-64, a selective cysteine protease inhibitor, but was completely prevented by AEBSF, a specific serine protease inhibitor. In addition, the potentiating effect of HDM on capsaicin-evoked Ca(2+) transient was absent in the pulmonary sensory neurons isolated from protease-activated receptor-2 (PAR(2)) knockout mice. Furthermore, the sensitizing effect of HDM was completely abolished by U73122, a phosholipase C inhibitor, or chelerythrine, a protein kinase C inhibitor. In summary, our results demonstrate that HDM, mainly through its serine protease activity, potentiates capsaicin-evoked Ca(2+) transient in mouse pulmonary sensory neurons via the activation of PAR(2) and the phosholipase C-protein kinase C intracellular transduction cascade.

Stimulatory Effect of 5‐hydroxytryptamine (5‐HT) on Rat Capsaicin‐sensitive Vagal Pulmonary Sensory Neurons via Activation of 5‐HT 3 Receptors

We have previously reported that 5-HT exerted a stimulatory effect on pulmonary vagal C-fiber afferents in anesthetized, artificially ventilated SD rats. However, whether this stimulatory effect of 5-HT is caused by a direct action on these afferents remains unknown. In the present study, the effect of 5-HT on the change in intracellular calcium concentration was determined in isolated nodose and jugular ganglion neurons innervating the rat lungs. Our results showed that: 1) 5-HT induced intensive calcium transients in the vagal pulmonary neurons in a dose-dependent manner. The highest concentration (3 μM) of 5-HT activated 66.7% (18/27) of the capsaicin-sensitive pulmonary neurons. 2) The 5-HT-indcued response was totally abolished by pretreatment of tropisetron (1 nM), a selective antagonist of 5-HT3 receptors. 3) Our whole-cell perforated patch-clamp recording showed that 5-HT triggered an inward current in the capsaicin-sensitive pulmonary neurons, and the response was abolished after pretreatment with tropisetron. In conclusion, 5-HT activated pulmonary C fibers in vivo and isolated pulmonary capsaicin-sensitive neurons in vitro, and probably resulted from its direct action on 5-HT3 receptors expressed in these neurons. (NIH HL 96914, fellowship NSC99-2918-I-038-001)