Pulmonary C-fiber Research Articles

Vagal-α7nAChR signaling attenuates allergic asthma responses and facilitates asthma tolerance by regulating inflammatory group 2 innate lymphoid cells.

Disorders of immune tolerance may lead to allergic asthma. Group 2 innate lymphoid cells (ILC2s) and inflammatory ILC2s (iILC2s) are key players in asthma. The vagus nerve innervating the airways releases acetylcholine or neuropeptides (i.e. calcitonin gene-related peptide) via pulmonary C-fibers (PCFs), which could regulate ILC2 activity upon binding the α7 nicotinic acetylcholine receptor (α7nAChR, coded by Chrna7) or neuropeptide receptors. Whether and how α7nAChR and PCFs regulate asthma and the formation of asthma tolerance via ILC2s or iILC2s are poorly understood. We used vagotomized, PCF degeneration and Chrna7 knockout mice to investigate ovalbumin (OVA)-induced asthma and oral OVA feeding-induced asthma tolerance. Our results revealed that vagotomy could generally suppress lung ILC2s and iILC2s, which mitigated allergic asthma responses but disrupted asthmatic tolerance. Removal of neuropeptides by PCF degeneration also reduced lung ILC2s and iILC2s, attenuating asthma responses, but did not affect asthma tolerance. In comparison, deletion of Chrna7 increased resident ILC2s and trafficking iILC2s in the lung, worsened allergic inflammation and disrupted oral tolerance. Mechanistically, deletion of Chrna7 in asthma-tolerant conditions upregulated T helper 2 cytokine- (Il4, Il13 and Il25) and sphingosine-1-phosphate (S1P)-related genes (S1pr1 and Sphk1). Blockade of S1P reduced iILC2 recruitment into asthmatic lungs. Our work is the first to demonstrate that vagal-α7nAChR signaling engaging with iILC2s and S1P not only alleviates asthma but also facilitates asthma tolerance. These findings may provide a novel therapeutic target for attenuating asthma by enhancing asthmatic tolerance.

Role of TRP channels in Gq-coupled protease-activated receptor 1-mediated activation of mouse nodose pulmonary C-fibers.

We evaluated the mechanisms underlying protease-activated receptor 1 (PAR1)-mediated activation of nodose C-fibers in mouse lungs. The PAR1-induced action potential discharge at the terminals was strongly inhibited in phospholipase C-β3 (PLCβ3)-deficient animals. At the level of the cell soma, PAR1 activation led to an increase in cytosolic calcium that was largely inhibited by transient receptor potential (TRP) A1 antagonism. Patch-clamp recordings, however, revealed that neither TRPA1 nor TRPV1 or any other ruthenium red-sensitive ion channels are required for the PAR1-mediated inward current or membrane depolarization in isolated nodose neurons. Consistent with these findings, PAR1-mediated action potential discharge in mouse lung nodose C-fiber terminals was unaltered in Trpa1/Trpv1 double-knockout animals and Trpc3/Trpc6 double-knockout animals. The activation of the C-fibers was also not inhibited by ruthenium red at concentrations that blocked TRPV1- and TRPA1-dependent responses. The biophysical data show that PAR1/Gq-mediated activation of nodose C-fibers may involve multiple ion channels downstream from PLCβ3 activation. TRPA1 is an ion channel that participates in PAR1/Gq-mediated elevation in intracellular calcium. There is little evidence, however, that TRPA1, TRPV1, TRPC3, TRPC6, or other ruthenium red-sensitive TRP channels are required for PAR1/Gq-PLCβ3-mediated membrane depolarization and action potential discharge in bronchopulmonary nodose C-fibers in the mouse.

Differential Activation of TRPA1 by Diesel Exhaust Particles: Relationships between Chemical Composition, Potency, and Lung Toxicity.

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.

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.

Pulmonary C-fiber degeneration downregulates IFN-γ receptor 1 via IFN-α induction to attenuate RSV-induced airway hyperresponsiveness

Respiratory syncytial virus (RSV) is a leading cause of respiratory infection in infants. Unfortunately, no effective vaccine or treatment against RSV is currently available. Pulmonary C-fibers (PCFs) are critical for regulating pulmonary inflammation and airway hyperresponsiveness (AHR). We previously reported that IFN-γ partially mediated RSV-induced airway disorders. In this study, we found that PCF degeneration alleviated RSV-induced airway inflammation, especially AHR by downregulating IFN-γ receptor 1 (IFNGR1), but had no effect on IFN-γ induction. In contrast, PCF degeneration actually increased IFN-α/β levels, as were the levels of STAT1 and phosphorylated STAT1 (pSTAT1). Exogenous IFN-α treatment induced STAT1 activation and downregulated IFNGR1 expression. These results suggest that PCFs affect IFNGR1 expression by inducing IFN-α to regulate IFN-γ-mediated airway inflammation and AHR. Thus, targeting PCFs activation may help control RSV-induced airway disorders, especially AHR, even with the presence of inflammation.

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.

Pulmonary C Fibers Modulate MMP-12 Production via PAR2 and Are Involved in the Long-Term Airway Inflammation and Airway Hyperresponsiveness Induced by Respiratory Syncytial Virus Infection.

Children with acute respiratory syncytial virus (RSV) infection often develop sequelae of persistent airway inflammation and wheezing. Pulmonary C fibers (PCFs) are involved in the generation of airway inflammation and resistance; however, their role in persistent airway diseases after RSV is unexplored. Here, we elucidated the pathogenesis of PCF activation in RSV-induced persistent airway disorders. PCF-degenerated and intact mice were used in the current study. Airway inflammation and airway resistance were evaluated. MMP408 and FSLLRY-NH2 were the selective antagonists for MMP-12 and PAR2, respectively, to investigate the roles of MMP-12 and PAR2 in PCFs mediating airway diseases. As a result, PCF degeneration significantly reduced the following responses to RSV infection: augmenting of inflammatory cells, especially macrophages, and infiltrating of inflammatory cells in lung tissues; specific airway resistance (sRaw) response to methacholine; and upregulation of MMP-12 and PAR2 expression. Moreover, the inhibition of MMP-12 reduced the total number of cells and macrophages in bronchiolar lavage fluid (BALF), as well infiltrating inflammatory cells, and decreased the sRaw response to methacholine. In addition, PAR2 was upregulated especially at the later stage of RSV infection. Downregulation of PAR2 ameliorated airway inflammation and resistance following RSV infection and suppressed the level of MMP-12. In all, the results suggest that PCF involvement in long-term airway inflammation and airway hyperresponsiveness occurred at least partially via modulating MMP-12, and the activation of PAR2 might be related to PCF-modulated MMP-12 production. Our initial findings indicated that the inhibition of PCF activity would be targeted therapeutically for virus infection-induced long-term airway disorders. The current study is critical to understanding that PCFs are involved in long-term airway inflammation and airway resistance after RSV infection through mediating MMP-12 production via PAR2, indicating that the inhibition of PCF activity can be targeted therapeutically for virus infection-induced long-term airway disorders.

Increased expression of macrophage migration inhibitory factor in the nucleus of solitary tract attenuates the renovascular hypertension

We investigatedwhether hemorrhagic hypotension (HH) altered the sensitivity of vagal pulmonary C-fibers in this study. The fiber activity (FA) of single vagal pulmonary C-fiber was continuously recorded in anesthetized rats before, during, and after HH was induced by bleeding from the femoral arterial catheter into a blood reservoir and lowering the mean systemic arterial blood pressure (MSAP) to ~40 mmHg for 20 min. Our results showed: 1)AfterMSAP reached a steady state of HH, the peak FA response to intravenous injection of capsaicin was elevated by ~5 folds. The enhanced C-fiber sensitivity continued to increase during HH and sustained even after MSAP returned to baseline during the recovery, but slowly returned to control ~20 min later. 2) Responses of FA to intravenous injections of other chemical stimulants of pulmonary Cfibers (phenylbiguanide, lactic acid and adenosine) and a constantpressure lung hyperinflation were all significantly potentiated by HH. 3) Infusion of sodium bicarbonate alleviated the systemic acidosis during HH, and it also attenuated, but did not completely prevent the HHinduced C-fiber hypersensitivity. In conclusion, the pulmonary C-fiber sensitivity was elevated during HH, probably caused by the endogenous release of chemical substances (e.g., lactic acid) that were produced by tissue ischemia during HH. This enhanced C-fiber sensitivity may heighten the pulmonary protective reflexes mediated through these afferents (e.g., J reflex) during hemorrhage when the body is more susceptible to other hazardous insults and pathophysiological stresses. (Supported in part by NHLBI and DoD grants)

Hypersensitivity of vagal pulmonary C-fibers is induced by hemorrhagic hypotension in anesthetized rats

Hypersensitivity of vagal pulmonary C-fibers is induced by hemorrhagic hypotension in anesthetized rats

Prenatal nicotinic exposure upregulates pulmonary C-fiber NK1R expression to prolong pulmonary C-fiber-mediated apneic response

Prenatal nicotinic exposure (PNE) prolongs bronchopulmonary C-fiber (PCF)-mediated apneic response to intra-atrial bolus injection of capsaicin in rat pups. The relevant mechanisms remain unclear. Pulmonary substance P and adenosine and their receptors (neurokinin-A receptor, NK1R and ADA1 receptor, ADA1R) and transient receptor potential cation channel subfamily V member 1 (TRPV1) expressed on PCFs are critical for PCF sensitization and/or activation. Here, we compared substance P and adenosine in BALF and NK1R, ADA1R, and TRPV1 expression in the nodose/jugular (N/J) ganglia (vagal pulmonary C-neurons retrogradely labeled) between Ctrl and PNE pups. We found that PNE failed to change BALF substance P and adenosine content, but significantly upregulated both mRNA and protein TRPV1 and NK1R in the N/J ganglia and only NK1R mRNA in pulmonary C-neurons. To define the role of NK1R in the PNE-induced PCF sensitization, the apneic response to capsaicin (i.v.) without or with pretreatment of SR140333 (a peripheral and selective NK1R antagonist) was compared and the prolonged apnea by PNE significantly shortened by SR140333. To clarify if the PNE-evoked responses depended on action of nicotinic acetylcholine receptors (nAChRs), particularly α7nAChR, mecamylamine or methyllycaconitine (a general nAChR or a selective α7nAChR antagonist) was administrated via another mini-pump over the PNE period. Mecamylamine or methyllycaconitine eliminated the PNE-evoked mRNA and protein responses. Our data suggest that PNE is able to elevate PCF NK1R expression via activation of nAChRs, especially α7nAChR, which likely contributes to sensitize PCFs and prolong the PCF-mediated apneic response to capsaicin.

Hypersensitivity of vagal pulmonary C-fibers induced by increasing airway temperature in ovalbumin-sensitized rats.

Our recent study has shown that hyperventilation of humidified warm air (HWA) triggered cough and reflex bronchoconstriction in patients with mild asthma. We suggested that a sensitizing effect on bronchopulmonary C-fibers by increasing airway temperature was involved, but direct evidence was lacking. This study was carried out to test the hypothesis that HWA enhances the pulmonary C-fiber sensitivity in Brown-Norway rats sensitized with ovalbumin (Ova). In anesthetized rats, isocapnic hyperventilation of HWA for 3 min rapidly elevated airway temperature to a steady state of 41.7°C. Immediately after the HWA challenge, the baseline fiber activity (FA) of pulmonary C-fibers was markedly elevated in sensitized rats, but not in control rats. Furthermore, the response of pulmonary C-fibers to right atrial injection of capsaicin in sensitized rats was significantly higher than control rats before the HWA challenge, and the response to capsaicin was further amplified after HWA in sensitized rats (ΔFA = 4.51 ± 1.02 imp/s before, and 9.26 ± 1.74 imp/s after the HWA challenge). A similar pattern of the HWA-induced potentiation of the FA response to phenylbiguanide, another chemical stimulant of C-fibers, was also found in sensitized rats. These results clearly demonstrated that increasing airway temperature significantly elevated both the baseline activity and responses to chemical stimuli of pulmonary C-fibers in Ova-sensitized rats. In conclusion, this study supports the hypothesis that the increased excitability of these afferents may have contributed to the cough and reflex bronchoconstriction evoked by hyperventilation of HWA in patients with asthma.

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.

Pulmonary C‐Fibers (PCFs) Are Essential for Airway Inflammation (AI) and Hyperreactivity (AHR) Induced by Respiratory Syncytial Virus (RSV) Infection

RSV infection causes acute AI and AHR with the peak response occurring at ~5th day postinfection (PID 5) in humans and animals. Owing to the critical role of PCFs in regulating pulmonary inflammation and airway resistance, we tested the contribution of PCFs to these RSV‐induced disorders. These RSV‐induced disorders are reportedly dependent on increased IFN‐γ in our previous study. IFN‐γ could activate C‐fibers and the latter affect the production of the former. Thus, we asked whether PCFs were required in IFN‐γ upregulation by RSV and the IFN‐γ‐mediated AI and AHR. RSV and mock were instilled intranasally in PCF‐intact (CON) and PCF‐degenerated (KPCF) mice. On PID 5, we measured specific airway resistance (sRaw) in awake state and collected lung tissues after euthanasia for detecting BALF inflammatory cells, the lung viral titration, substance P (SP), IFN‐γ and its transcription factor (T‐bet) gene expressions. Compared with CON, KPCF mice showed significant decreases in: (1) weight loss and RSV titer in the lungs; (2) total cells and macrophages in BALF with little change in lymphocytes; (3) inflammatory cells infiltrated in the lungs; (4) the sRaw response to aerosol of methacholine (3.125 ‐ 50 mg/ml); and (5) SP. In addition, the augmented IFN‐γ and T‐bet by RSV infection was not significantly different between the two groups of mice. However, recombinant IFN‐γ (ip) induced AHR in the CON, but not KPCF mice. Our results suggest that PCFs play an important role in RSV replication, the RSV‐induced AI and AHR. Moreover, PCFs were required for IFN‐γ‐promoted AI and AHR (Supported by HL119683).

INHALATION OF HYDROGEN SULFIDE (H2S) AUGMENTS THE PULMONARY REFLEXES

We have previously reported that H2S exerted a sensitizing effect on pulmonary vagal C‐fiber afferents in anesthetized, artificially ventilated SD rats. However, whether the sensitizing effect of H2S in turn enhances pulmonary reflexes remains unknown. In anesthetized, spontaneously breathing rats, inhalation of aerosolized sodium hydrosulfide (NaHS, a donor of H2S) caused no significant changes in the baseline breathing pattern. However, the pulmonary chemoreflexes, characterized by apnea, bradycardia and hypotension, triggered by right atrial injection of capsaicin or adenosine, were potentiated after the NaHS inhalation. This potentiating effect was eliminated by pretreatment with perineural capsaicin (a selective blockade of the conduction of vagal C‐fibers) or HC‐030031 (an antagonist of TRPA1 receptors), but not affected by pretreatment with perineural sham surgery or HC‐030031 vehicle. Furthermore, in awake, restrained guinea pigs, the cough reflexes elicited by inhalation of aerosolized capsaicin were also potentiated after the NaHS inhalation. In conclusion, H2S enhanced pulmonary reflexes, and probably resulted from its action on pulmonary C‐fibers. (MOST 103‐2320‐B‐038‐014‐)

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.

Differential Inhibition of Bilateral Phrenic Bursting following Pulmonary Chemoreflex in Rats with Chronic Cervical Spinal Hemisection

High cervical spinal injury usually interrupts bulbospinal pathways and results in inactivation of phrenic bursting ipsilateral to the lesion. The ipsilateral phrenic activity can partially recover over weeks to months post‐injury due to activation of the latent crossed phrenic pathway. Ipsilateral phrenic nerve exhibits a greater capacity to increase activity during respiratory challenges than the contralateral phrenic nerve. However, whether bilateral phrenic nerves show a differential response to respiratory inhibitory inputs is unclear. Accordingly, present study was designed to examine bilateral phrenic bursting in response to capsaicin‐induced pulmonary chemoreflex, a robust respiratory inhibitory stimulus. Bilateral phrenic nerve activity were recorded in anesthetized and mechanically‐ventilated adult rats at 8‐9 weeks post‐C2 hemisection (C2Hx) or C2 laminectomy. Intra‐jugular capsaicin (1.5 µg/kg) injection was used to activate pulmonary C‐fibers to evoke the pulmonary chemoreflex. Present results showed capsaicin‐induced prolongation of expiratory duration (i.e., apnea) was significantly attenuated in C2Hx animals. However, ipsilateral phrenic activity was robustly inhibited after capsaicin treatment compared to contralateral phrenic nerve of C2Hx animals or ipsilateral phrenic nerve of uninjured animals. In addition, discharge onset of ipsilateral phrenic bursting of C2Hx animals is significantly delayed after capsaicin delivery. These results suggest that phrenic motor output is more susceptible to inhibitory inputs after cervical hemisection. Moreover, central (i.e., apnea) and spinal (i.e., inhibition of phrenic bursting) component of the pulmonary chemoreflex were differentially modulated during chronic injury phase.

Hemorrhagic hypotension-induced hypersensitivity of vagal pulmonary C-fibers to chemical stimulation and lung inflation in anesthetized rats.

This study was carried out to investigate whether hemorrhagic hypotension (HH) altered the sensitivity of vagal pulmonary C-fibers. The fiber activity (FA) of single vagal pulmonary C-fiber was continuously recorded in anesthetized rats before, during, and after HH was induced by bleeding from the femoral arterial catheter into a blood reservoir and lowering the mean systemic arterial pressure (MSAP) to ∼40 mmHg for 20 min. Our results showed the following. First, after MSAP reached a steady state of HH, the peak FA response to intravenous injection of capsaicin was elevated by approximately fivefold. The enhanced C-fiber sensitivity continued to increase during HH and sustained even after MSAP returned to baseline during the recovery, but slowly returned to control ∼20 min later. Second, responses of FA to intravenous injections of other chemical stimulants of pulmonary C-fibers (phenylbiguanide, lactic acid, and adenosine) and a constant-pressure lung hyperinflation were all significantly potentiated by HH. Third, infusion of sodium bicarbonate alleviated the systemic acidosis during HH, and it also attenuated, but did not completely prevent, the HH-induced C-fiber hypersensitivity. In conclusion, the pulmonary C-fiber sensitivity was elevated during HH, probably caused by the endogenous release of chemical substances (e.g., lactic acid) that were produced by tissue ischemia during HH. This enhanced C-fiber sensitivity may heighten the pulmonary protective reflexes mediated through these afferents (e.g., cough, J reflex) during hemorrhage when the body is more susceptible to other hazardous insults and pathophysiological stresses.

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.