Stimulatory effect of methylglyoxal on capsaicin-sensitive lung vagal afferents in rats: role of TRPA1.

Methylglyoxal (MG), a reactive metabolic byproduct of glycolysis, is a causative of painful diabetic neuropathy. Patients with diabetes are associated with more frequent severe asthma exacerbation. Stimulation of capsaicin-sensitive lung vagal (CSLV) afferents may contribute to the pathogenesis of hyperreactive airway diseases such as asthma. However, the possibility of the stimulatory effect of MG on CSLV afferents and the underlying mechanisms remain unknown. Our results showed that intravenous injection of MG (25 mg/kg, MG25) in anesthetized, spontaneously breathing rats elicited pulmonary chemoreflexes characterized by apnea, bradycardia, and hypotension. The MG-induced apneic response was reproducible and dose dependent. MG25 no longer evoked these reflex responses after perineural capsaicin treatment of both cervical vagi to block C-fibers' conduction, suggesting that the reflexes were mediated through the stimulation of CSLV afferents. Pretreatment with HC030031 [an antagonist of transient receptor potential ankyrin subtype 1 protein (TRPA1)] or AP18 (another TRPA1 antagonist), but not their vehicle, markedly attenuated the apneic response induced by MG25. Consistently, electrophysiological results showed that pretreatment with HC030031 largely attenuated the intense discharge in CSLV afferents induced by injection of MG25 in open-chest and artificially ventilated rats. In isolated CSLV neurons, the perfusion of MG evoked an abrupt and pronounced increase in calcium transients in a concentration-dependent manner. This stimulatory effect on CSLV neurons was also abolished by HC030031 treatment but not by its vehicle. In conclusion, these results suggest that MG exerts a stimulatory effect on CSLV afferents, inducing pulmonary chemoreflexes, and such stimulation is mediated through the TRPA1 activation.NEW & NOTEWORTHY Methylglyoxal (MG) is implicated in the development of painful diabetic neuropathy. A retrospective cohort study revealed an increased incidence of asthma exacerbations in patients with diabetes. This study demonstrated that elevated circulating MG levels stimulate capsaicin-sensitive lung vagal afferents via activation of TRPA1, which in turn triggers respiratory reflexes. These findings provide new information for understanding the pathogenic mechanism of diabetes-associated hyperreactive airway diseases and potential therapy.

Identifying vagal bronchopulmonary afferents mediating cough response to inhaled sulfur dioxide in mice.

Sulfur dioxide (SO2), a common environmental and industrial air pollutant, possesses a potent effect in eliciting cough reflex, but the primary type of airway sensory receptors involved in its tussive action has not been clearly identified. This study was carried out to determine the relative roles of three major types of vagal bronchopulmonary afferents [slowly adapting receptors (SARs), rapidly adapting receptors (RARs), and C-fibers] in regulating the cough response to inhaled SO2. Our results showed that inhalation of SO2 (300 or 600 ppm for 8 min) evoked an abrupt and intense stimulatory effect on bronchopulmonary C-fibers, which continued for the entire duration of inhalation challenge and returned toward the baseline in 1-2 min after resuming room air-breathing in anesthetized and mechanically ventilated mice. In stark contrast, the same SO2 inhalation challenge generated a distinct and consistent inhibitory effect on both SARs and phasic RARs; their phasic discharges synchronized with respiratory cycles during the baseline (breathing room air) began to decline progressively within 1-3 min after the onset of SO2 inhalation, ceased completely before termination of the 8-min inhalation challenge, and then slowly returned toward the baseline after >40 min. In a parallel study in awake mice, inhalation of SO2 at the same concentration and duration as that in the nerve recording experiments evoked cough responses in a pattern and time course similar to that observed in the C-fiber responses. Based on these results, we concluded that stimulation of vagal bronchopulmonary C-fibers is primarily responsible for triggering the cough response to inhaled SO2.NEW & NOTEWORTHY This study demonstrated that inhalation of a high concentration of sulfur dioxide, an irritant gas and common air pollutant, completely and reversibly inhibited the neural activities of both slowly adapting receptor and rapidly adapting receptor, two major types of mechanoreceptors in the lungs with their activities conducted by myelinated fibers. Furthermore, the results of this study suggested that stimulation of vagal bronchopulmonary C-fibers is primarily responsible for triggering the cough reflex responses to inhaled sulfur dioxide.

Hypersensitivity of Airway Reflexes Induced by Hydrogen Sulfide: Role of TRPA1 Receptors.

The activation of capsaicin-sensitive lung vagal (CSLV) afferents can elicit airway reflexes. Hypersensitivity of these afferents is known to contribute to the airway hypersensitivity during airway inflammation. Hydrogen sulfide (H2S) has been suggested as a potential therapeutic agent for airway hypersensitivity diseases, such as asthma, because of its relaxing effect on airway smooth muscle and anti-inflammatory effect. However, it is still unknown whether H2S affects airway reflexes. Our previous study demonstrated that exogenous application of H2S sensitized CSLV afferents and enhanced Ca2+ transients in CSLV neurons. The present study aimed to determine whether the H2S-induced sensitization leads to functional changes in airway reflexes and elevates the electrical excitability of the CSLV neurons. Our results showed that, first and foremost, in anesthetized, spontaneously breathing rats, the inhalation of aerosolized sodium hydrosulfide (NaHS, a donor of H2S; 5 mg/mL, 3 min) caused an enhancement in apneic response evoked by several stimulants of the CSLV afferents. This enhancement effect was found 5 min after NaHS inhalation and returned to control 30 min later. However, NaHS no longer enhanced the apneic response after perineural capsaicin treatment on both cervical vagi that blocked the conduction of CSLV fibers. Furthermore, the enhancing effect of NaHS on apneic response was totally abolished by pretreatment with intravenous HC-030031 (a TRPA1 antagonist; 8 mg/kg), whereas the potentiating effect was not affected by the pretreatment with the vehicle of HC-030031. We also found that intracerebroventricular infusion pretreated with HC-030031 failed to alter the potentiating effect of NaHS on the apneic response. Besides, the cough reflex elicited by capsaicin aerosol was enhanced by inhalation of NaHS in conscious guinea pigs. Nevertheless, this effect was entirely eliminated by pretreatment with HC-030031, not by its vehicle. Last but not least, voltage-clamp electrophysiological analysis of isolated rat CSLV neurons showed a similar pattern of potentiating effects of NaHS on capsaicin-induced inward current, and the involvement of TRPA1 receptors was also distinctly shown. In conclusion, these results suggest that H2S non-specifically enhances the airway reflex responses, at least in part, through action on the TRPA1 receptors expressed on the CSLV afferents. Therefore, H2S should be used with caution when applying for therapeutic purposes in airway hypersensitivity diseases.

Involvement of Capsaicin-Sensitive Lung Vagal Neurons and TRPA1 Receptors in Airway Hypersensitivity Induced by 1,3-ß-D-Glucan in Anesthetized Rats.

Airway exposure to 1,3-ß-D-glucan (ß-glucan), an essential component of the cell wall of several pathogenic fungi, causes various adverse responses, such as pulmonary inflammation and airway hypersensitivity. The former response has been intensively investigated; however, the mechanism underlying ß-glucan-induced airway hypersensitivity is unknown. Capsaicin-sensitive lung vagal (CSLV) afferents are very chemosensitive and stimulated by various insults to the lungs. Activation of CSLV afferents triggers several airway reflexes, such as cough. Furthermore, the sensitization of these afferents is known to contribute to the airway hypersensitivity during pulmonary inflammation. This study was carried out to determine whether ß-glucan induces airway hypersensitivity and the role of the CSLV neurons in this hypersensitivity. Our results showed that the intratracheal instillation of ß-glucan caused not only a distinctly irregular pattern in baseline breathing, but also induced a marked enhancement in the pulmonary chemoreflex responses to capsaicin in anesthetized, spontaneously breathing rats. The potentiating effect of ß-glucan was found 45 min later and persisted at 90 min. However, ß-glucan no longer caused the irregular baseline breathing and the potentiating of pulmonary chemoreflex responses after treatment with perineural capsaicin treatment that blocked the conduction of CSLV fibers. Besides, the potentiating effect of ß-glucan on pulmonary chemoreflex responses was significantly attenuated by N-acetyl-L-cysteine (a ROS scavenger), HC-030031 (a TRPA1 antagonist), and Laminarin (a Dectin-1 antagonist). A combination of Laminarin and HC-030031 further reduced the ß-glucan-induced effect. Indeed, our fiber activity results showed that the baseline fiber activity and the sensitivity of CSLV afferents were markedly elevated by ß-glucan instillation, with a similar timeframe in anesthetized, artificially ventilated rats. Moreover, this effect was reduced by treatment with HC-030031. In isolated rat CSLV neurons, the ß-glucan perfusion caused a similar pattern of potentiating effects on capsaicin-induced Ca2+ transients, and ß-glucan-induced sensitization was abolished by Laminarin pretreatment. Furthermore, the immunofluorescence results showed that there was a co-localization of TRPV1 and Dectin-1 expression in the DiI-labeled lung vagal neurons. These results suggest that CSLV afferents play a vital role in the airway hypersensitivity elicited by airway exposure to ß-glucan. The TRPA1 and Dectin-1 receptors appear to be primarily responsible for generating ß-glucan-induced airway hypersensitivity.

Release of ATP in the lung evoked by inhalation of irritant gases in rats.

Adenosine triphosphate (ATP) can be released into the extracellular milieu from various types of cells in response to a wide range of physical or chemical stresses. In the respiratory tract, extracellular ATP is recognized as an important signal molecule and trigger of airway inflammation. Chlorine (Cl2), sulfur dioxide (SO2), and ammonia (NH3) are potent irritant gases and common industrial air pollutants due to their widespread uses as chemical agents. This study was carried out to determine if acute inhalation challenges of these irritant gases, at the concentration and duration simulating the accidental exposures to these chemical gases in industrial operations, triggered the release of ATP in the rat respiratory tract; and if so, whether the level of ATP in bronchoalveolar lavage fluid (BALF) evoked by inhalation challenge of a given irritant gas was elevated by chronic allergic airway inflammation. Our results showed: 1) inhalation of these irritant gases caused significant increases in the ATP level in BALF, and the magnitude of evoked ATP release was in the order of Cl2 > SO2 > NH3. 2) Chronic airway inflammation induced by ovalbumin-sensitization markedly elevated the ATP level in BALF during baseline (breathing room air) but did not potentiate the release of ATP in the lung triggered by inhalation challenges of these irritant gases. These findings suggested a possible involvement of the ATP release in the lung in the regulation of overall airway responses to acute inhalation of irritant gases and the pathogenesis of chronic allergic airway inflammation.NEW & NOTEWORTHY Extracellular adenosine triphosphate (ATP) is a contributing factor and signaling molecule of airway inflammation. This study demonstrated for the first time that the ATP release in the lung was markedly elevated after acute inhalation challenges of three common industrial air pollutants; the order of the response magnitude was chlorine > sulfur dioxide > ammonia. These findings provided new information and improved our understanding of the adverse pulmonary effects caused by accidental inhalation exposures to these irritant gases.

Inhibitory effect of sulfur dioxide inhalation on Hering-Breuer inflation reflex in mice: role of voltage-gated potassium channels.

Slowly adapting receptors (SARs), vagal mechanosensitive receptors located in the lung, play an important role in regulating the breathing pattern and Hering-Breuer inflation reflex (HBIR). Inhalation of high concentration of sulfur dioxide (SO2), a common environmental and occupational air pollutant, has been shown to selectively block the SAR activity in rabbits, but the mechanism underlying this inhibitory effect remained a mystery. We carried out this study to determine if inhalation of SO2 can inhibit the HBIR and change the eupneic breathing pattern, and to investigate further a possible involvement of voltage-gated K+ channels in the inhibitory effect of SO2 on these vagal reflex-mediated responses. Our results showed 1) inhalation of SO2 (600 ppm; 8 min) consistently abolished both the phasic activity of SARs and their response to lung inflation in anesthetized, artificially ventilated mice, 2) inhalation of SO2 generated a distinct inhibitory effect on the HBIR and induced slow deep breathing in anesthetized, spontaneously breathing mice, and these effects were reversible and reproducible in the same animals, 3) This inhibitory effect of SO2 was blocked by pretreatment with 4-aminopyridine (4-AP), a nonselective blocker of voltage-gated K+ channel, and unaffected by pretreatment with its vehicle. In conclusion, this study suggests that this inhibitory effect on the baseline breathing pattern and the HBIR response was primarily mediated through the SO2-induced activation of voltage-gated K+ channels located in the vagal bronchopulmonary SAR neurons.NEW & NOTEWORTHY This study demonstrated that inhaled sulfur dioxide completely and reversibly abolished the activity of vagal bronchopulmonary slowly adapting receptors, significantly inhibited the apneic response to lung inflation, and induced slow deep breathing in anesthetized mice. More importantly, our results further suggested that this inhibitory effect was mediated through an action of sulfur dioxide and its derivatives on the voltage-gated potassium channels expressed in the slowly adapting receptor sensory neurons innervating the lung.

Premorbid use of selective beta-blockers improves sepsis incidence and course: Human cohort and animal model studies.

Introduction: Beta-blockers are widely prescribed to manage hypertension and cardiovascular diseases and have been suggested as an attractive therapy to improve the prognosis of sepsis. Herein, we investigated the potential benefits of premorbid selective beta-blocker use in sepsis with a real-world database and explored the underlying mechanism by in vivo and in vitro experiments. Methods: A total of 64,070 sepsis patients and 64,070 matched controls who were prescribed at least one anti-hypertensive drug for more than 300 days within 1 year were selected for the nested case-control study. Female C57BL/6 J mice and THP-1 cells stimulated with lipopolysaccharide (LPS) were used for studying systemic responses during sepsis to validate our clinical findings. Results: The risk of sepsis was lower in current selective beta-blocker users than in non-users (adjusted OR (aOR), 0.842; 95% CI, 0.755-0.939), and in recent users than in non-users (aOR, 0.773; 95% CI, 0.737-0.810). A mean daily dose of ≥0.5 DDD was associated with a lower risk of sepsis (aOR, 0.7; 95% CI, 0.676-0.725). Metoprolol, atenolol, and bisoprolol users had lower risk of sepsis than non-users. In a LPS-induced sepsis mouse model, mice pre-fed with atenolol had significantly reduced mortality. While atenolol had some mild effects on LPS-induced release of inflammatory cytokines in septic mice, it significantly reduced serum soluble PD-L1 levels. Notably, atenolol treatment reversed the negative correlation of sPD-L1 with inflammatory cytokines in septic mice. Moreover, atenolol markedly downregulated the PD-L1 expression on LPS-stimulated THP-1 monocytes/macrophages via targeting ROS-induced NF-κB and STAT3 activation. Conclusion: Atenolol pretreatment can reduce sepsis mortality in mice, and in vivo and in vitro studies of PD-L1 expression suggest a role for atenolol in the modulation of immune homeostasis. These findings may contribute to the reduced incidence of sepsis in hypertensive patients with premorbid treatment with selective beta-blockers, especially atenolol.

Airway Exposure to 1,3-Beta-d-Glucan Induces Airway Hyperresponsiveness in Guinea Pigs.

1,3-Beta-d-glucan (ß-glucan) is a component of mold cell walls and is frequently found in fungi and house dust mites. The studies of ß-glucan are inconsistent, although it has been implicated in airway adverse responses. This study was carried out to determine whether airway hyperresponsiveness was seen 24 h after airway exposure to ß-glucan in guinea pigs. Two matching guinea pigs were exposed intratracheally to either ß-glucan or its vehicle. Twenty-four hours after intratracheal instillation, there was no difference between these two groups in the baseline of the total pulmonary resistance (R L), dynamic lung compliance (C dyn), arterial blood pressure, and heart rate. In contrast, the responses of R L to capsaicin injection were significantly increased in ß-glucan animals; capsaicin at the same dose of 3.2 µg/kg increased R L by 184% in vehicle animals and by 400% in ß-glucan animals. The effective dose 200% to capsaicin injection was lower in the ß-glucan animals. Furthermore, the increases in R L were partially reduced after transient lung hyperinflation to recruit the occluding airways; however, the R L induced by capsaicin injection after lung hyperinflation was significantly larger than the baseline in ß-glucan animals; also, the lung wet-to-dry ratio in capsaicin-injected animals was augmented in the ß-glucan group. Moreover, the airway hyperresponsiveness was accompanied by increases in neutrophils in the bronchoalveolar lavage fluid in the ß-glucan animals. Furthermore, the levels of substance P and the calcitonin gene-related peptide in the bronchoalveolar lavage fluid collected after capsaicin injection were increased in ß-glucan animals. We provide definitive evidence that ß-glucan can induce airway hyperresponsiveness in guinea pigs, and the neuropeptide releases play an important role in this airway hyperresponsiveness.