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.

Desmethylclomipramine triggers mitochondrial damage and death in TGF-ß-induced mesenchymal type of A549 cells.

Lung cancer is the leading cause of cancer deaths, where the metastasis often causes chemodrug resistance and leads to recurrence after treatment. Desmethylclomipramine (DCMI), a bioactive metabolite of clomipramine, shows the therapeutic efficacy with antidepressive agency as well as potential cytostatic effects on lung cancer cells. Here, we demonstrated that DCMI effectively caused transforming growth factor (TGF)-ß1-mediated mesenchymal type of A549 cells to undergo mitochondrial death via myeloid cell leukemia-1 (Mcl-1) suppression and activation of truncated Bid (tBid). TGF-ß1 induced epithelial mesenchymal transition in A549 cells with the increase of fibronectin and decrease of E-cadherin, the activation of Akt/glycogen synthase kinase-3ß (GSK-ß)/Mcl-1 axis, and the hypo-responsiveness to cisplatin. DCMI initiated a dose-dependent cytotoxicity on TGF-ß1-mediated mesenchymal type of A549 cells through inactivating Akt/GSK-ß/Mcl-1 axis, in which mitochondria instability and caspase-9/3 activation also occurred concurrently. Pharmacological inhibition of caspase-8 and cathepsin B partly reversed tBid expression and mitochondrial damage to further attenuate DCMI-mediated cytotoxicity. Additionally, DCMI presented partial therapeutic effects in treating mesenchymal type of A549 tumor bearing nude mice through an acceleration of cancer cell death. Taken together, DCMI exerts antitumor effects via initiating the mechanisms of Akt/GSK-ß/Mcl-1 inactivation and cathepsin B/caspase-8-regulated mitochondrial death, which suggests its potential role in mesenchymal type of cancer cell therapy.

Endodontic Radiopacifying Application of Barium Titanate Prepared through a Combination of Mechanical Milling and Heat Treatment.

Mineral trioxide aggregates (MTA) are commonly used as endodontic filling materials but suffer from a long setting time and tooth discoloration. In the present study, the feasibility of using barium titanate (BTO) for discoloration and a calcium chloride (CaCl2) solution to shorten the setting time was investigated. BTO powder was prepared using high-energy ball milling for 3 h, followed by sintering at 700-1300 °C for 2 h. X-ray diffraction was used to examine the crystallinity and crystalline size of the as-milled and heat-treated powders. MTA-like cements were then prepared using 20-40 wt.% BTO as a radiopacifier and solidified using a 0-30% CaCl2 solution. The corresponding radiopacity, diametral tensile strength (DTS), initial and final setting times, and discoloration performance were examined. The experimental results showed that for the BTO powder prepared using a combination of mechanical milling and heat treatment, the crystallinity and crystalline size increased with the increasing sintering temperature. The BTO sintered at 1300 °C (i.e., BTO-13) exhibited the best radiopacity and DTS. The MTA-like cement supplemented with 30% BTO-13 and solidified with a 10% CaCl2 solution exhibited a radiopacity of 3.68 ± 0.24 mmAl and a DTS of 2.54 ± 0.28 MPa, respectively. In the accelerated discoloration examination using UV irradiation, the color difference was less than 1.6 and significantly lower than the clinically perceptible level (3.7). This novel MTA exhibiting a superior color stability, shortened setting time, and excellent biocompatibility has potential for use in endodontic applications.

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.

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.

Mirabegron, a ß3­adrenoreceptor agonist, regulates right and left atrial arrhythmogenesis differently.

Mirabegron increases atrial fibrillation (AF) risk. The left atrium (LA) is the most critical 'substrate' for AF and has higher arrhythmogenesis compared with the right atrium (RA). The present study aimed to investigate the electrophysiological and arrhythmogenic effects of mirabegron on the LA and RA and clarify the potential underlying mechanisms. Conventional microelectrodes, a whole-cell patch clamp and a confocal microscope were used in rabbit LA and RA preparations or single LA and RA myocytes before and after mirabegron administration with or without cotreatment with KT5823 [a cyclic adenosine monophosphate (cAMP)-dependent protein kinase inhibitor]. The baseline action potential duration at repolarization extents of 20 and 50% (but not 90%) were shorter in the LA than in the RA. Mirabegron at 0.1 and 1 µM (but not 0.01 µM) reduced the action potential duration at repolarization extents of 20 and 50% in the LA and RA. Mirabegron (0.1 µM) increased the occurrence of tachypacing-induced burst firing in the LA but not in the RA, where it was suppressed by KT5823 (1 µM). Mirabegron (0.1 µM) increased the L-type Ca2+ current (ICa-L), ultrarapid component of delayed rectifier K+ current (IKur), Ca2+ transients and sarcoplasmic reticulum Ca2+ content but reduced transient outward K+ current (Ito) in the LA myocytes. However, mirabegron did not change the Na+ current and delayed rectifier K+ current in the LA myocytes. Moreover, pretreatment with KT5823 (1 µM) inhibited the effects of mirabegron on ICa-L, Ito and IKur in the LA myocytes. Furthermore, in the RA myocytes, mirabegron reduced ICa-L but did not change Ito. In conclusion, mirabegron differentially regulates electrophysiological characteristics in the LA and RA. Through the activation of the cAMP-dependent protein kinase pathway and induction of Ca2+ dysregulation, mirabegron may increase LA arrhythmogenesis, leading to increased AF risk.

Mechanisms Involved in the Stimulatory and Inhibitory Effects of 5-Hydroxytryptamine on Vagal Mechanosensitive Afferents in Rat Lung.

Mechanosensitive vagal afferents in the lung, rapidly and slowly adapting receptors (RARs and SARs, respectively), play an important role in eliciting the reflexes that regulate the normal airway function. A profound bronchoconstrictive effect of 5-hydroxytryptamine (5-HT) has been extensively reported in various animal species, but its influence on the SAR and RAR activity is not known. This study investigated the effect of 5-HT on these receptors, and the possible mechanisms involved. Single-fiber activities of these afferents were measured in anesthetized, open-chest, and mechanically ventilated rats. Our results showed that intravenous injection of 5-HT evoked a consistent and pronounced stimulation of phasic RARs. In contrast, 5-HT generated an inconsistent and paradoxical action on SARs: no effect in 29% (5 of 17) of the SARs; stimulation in 35% (6 of 17); and inhibition in the remainder. These responses of both RARs and SARs to 5-HT were reproducible and dose-dependent. After the injection of a high dose of 5-HT (16 µg/kg), the receptor responses slowly reached a peak (after ∼8 s) and returned toward the baseline in ∼20 s, accompanied by a consistent increase in total pulmonary resistance and a decrease in dynamic lung compliance in a temporal pattern very similar to the increased receptor activity. When these changes in lung mechanics induced by 5-HT were prevented by pretreatment with salbutamol, a ß2 adrenergic receptor agonist, the delayed responses of both RARs and SARs to 5-HT were also abolished, except that the immediate stimulatory effect on a subset of RARs, the silent RARs, was not affected. In conclusion, 5-HT generated a delayed stimulatory effect on RARs and a paradoxical effect on SARs, which resulted primarily from the 5-HT-induced changes in mechanical properties of the lung.

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.

Efficacy and safety of anti-interleukin-5 therapy in patients with chronic obstructive pulmonary disease: A meta-analysis of randomized, controlled trials.

BACKGROUND: Anti-interleukin-5 (IL-5) therapy has been proposed as a novel treatment option for patients with chronic obstructive pulmonary disease (COPD). However, its efficacy for preventing COPD exacerbation remains unclear. METHODS: A literature review was conducted to August 26th 2019. Only randomized controlled trials (RCTs) that investigated the clinical efficacy and adverse effects of anti-IL-5 therapy were included in the meta-analysis. The primary outcome was the risk of COPD exacerbation. RESULTS: A total of 3 articles containing 5 RCTs were included in the study. Overall, 2837 and 1442 patients received anti-IL-5 therapy (mepolizumab, n = 865; benralizumab, n = 1972) and placebo, respectively. In the pooled analysis, anti-IL-5 therapy was associated with a lower risk of COPD exacerbation compared with the placebo (rate ratio, 0.92; 95% CI, 0.86-0.97, I2 = 0%). In addition, no significant differences in the changes in SGRQ scores and FEV1 from baseline were found between the anti-IL-5 therapy and placebo (SGRQ, mean difference, -0.86, 95% CI, -1.92 - 0.19, I2 = 0%; FEV1, mean difference, 0.01, 95% CI, -0.01 - 0.03, I2 = 0%). Anti-IL-5 therapy had a similar risk of any adverse event (risk ratio, 1.02; 95% CI, 0.99-1.05), an event leading to treatment discontinuation (risk ratio, 1.04; 95% CI, 0.72-1.48) and any serious adverse events (risk ratio, 0.93; 95% CI, 0.85-1.01) when compared with the placebo. CONCLUSION: Anti-IL-5 therapy was associated with a lower rate of COPD exacerbation compared with placebo. In addition, anti-IL-5 therapy was well tolerated for COPD patients.

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.

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.

Atrial arrhythmogenesis in a rabbit model of chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) increases the risk of atrial fibrillation (AF), however, its arrhythmogenic mechanisms are unclear. This study investigated the effects of COPD on AF triggers (pulmonary veins, PVs) and substrates (atria), and their potential underlying mechanisms. Electrocardiographic, echocardiographic, and biochemical studies were conducted in control rabbits and rabbits with human leukocyte elastase (0.3 unit/kg)-induced COPD. Conventional microelectrode, Western blotting, and histological examinations were performed on PV, left atrium (LA), right atrium, and sinoatrial node (SAN) preparations from control rabbits and those with COPD. The rabbits with COPD had a higher incidence of atrial premature complexes, PV burst firing and delayed afterdepolarizations, higher sympathetic activity, larger LA, and faster PV spontaneous activity than did the control rabbits; but they exhibited a slower SAN beating rate. The LA of the rabbits with COPD had a shorter action potential duration and longer tachyarrhythmia induced by tachypacing (20 Hz) and isoproterenol (1 µM). Additionally, the rabbits with COPD had higher fibrosis in the PVs, LA, and SAN. H89 (10 µM), KN93 (1 µM), and KB-R7943 (10 µM) significantly suppressed burst firing and delayed afterdepolarizations in the PVs of the rabbits with COPD. Moreover, compared with the control rabbits, those with COPD had lower expression levels of the ß1 adrenergic receptor, Cav 1.2, and Na+/Ca2+ exchanger in the PVs; Cav 1.2 in the LA; and hyperpolarization-activated cyclic nucleotide-gated K+ channel 4 in the SAN. COPD increases atrial arrhythmogenesis by modulating the distinctive electrophysiological characteristics of the PVs, LA, and SAN.

Mechanisms underlying the stimulatory effect of inhaled sulfur dioxide on vagal bronchopulmonary C-fibres.

KEY POINTS: Brief inhalation of SO2 of concentration >500 p.p.m. triggered a pronounced stimulatory effect on vagal bronchopulmonary C-fibres in anaesthetized rats. This stimulatory effect was drastically diminished by a pretreatment with NaHCO3 that raised the baseline arterial pH, suggesting a possible involvement of acidification of airway fluid and/or tissue generated by inhaled SO2 . The stimulation was completely abolished by pretreatment with antagonists of both acid-sensing ion channels and transient receptor potential vanilloid type-1 receptors, indicating that this effect was caused by acid activation of these cation channels expressed in airway sensory nerves. This conclusion was further supported by the results obtained from studies in isolated rat vagal bronchopulmonary sensory neurones and also in the cough response to SO2 inhalation challenge in awake mice. These results provide new insight into the underlying mechanism of harmful irritant effects in the respiratory tract caused by accidental exposure to a high concentration of SO2 . ABSTRACT: Inhalation of sulfur dioxide (SO2 ) triggers coughs and reflex bronchoconstriction, and stimulation of vagal bronchopulmonary C-fibres is primarily responsible. However, the mechanism underlying this stimulatory effect is not yet fully understood. In this study, we tested the hypothesis that the C-fibre stimulation was caused by SO2 -induced local tissue acidosis in the lung and airways. Single-unit activities of bronchopulmonary C-fibres in response to inhalation challenges of SO2 (500-1500 p.p.m., 10 breaths) were measured in anaesthetized rats. Inhalation of SO2 reproducibly induced a pronounced and sustained stimulation (lasting for 15-60 s) of pulmonary C-fibres in a concentration-dependent manner. This stimulatory effect was significantly attenuated by an increase in arterial pH generated by infusion of sodium bicarbonate (NaHCO3 ), and completely abrogated by a combined pretreatment with amiloride (an antagonist of acid-sensing ion channels, ASICs) and AMG8910 (a selective antagonist of the transient receptor potential vanilloid type-1 receptor, TRPV1). Furthermore, in isolated rat vagal pulmonary sensory neurones, perfusion of an aqueous solution of SO2 evoked a transient increase in the intracellular Ca2+ concentration; this response was also markedly diminished by a pretreatment with amiloride and AMG8910. In addition, inhalation of SO2 consistently evoked coughs in awake mice; responses were significantly smaller in TRPV1-/- mice than in wild-type mice, and almost completely abolished after a pretreatment with amiloride in TRPV1-/- mice. These results suggested that the stimulatory effect of inhaled SO2 on bronchopulmonary C-fibres was generated by acidification of fluid and/or tissue in the lung and airways, which activated both ASICs and TRPV1 expressed in these sensory nerves.

Stimulatory Effect of 5-Hydroxytryptamine (5-HT) on Rat Capsaicin-Sensitive Lung Vagal Sensory Neurons via Activation of 5-HT3 Receptors.

5-hydroxytryptamine (5-HT) is an inflammatory mediator known to be released in lung. Capsaicin-sensitive lung vagal (CSLV) afferents function as a primary sensor for detecting chemical stimuli and produce consequent reflexes during lung inflammation. To characterize the effect of 5-HT on CSLV afferents, responses of cardiorespiratory reflexes and single-unit C-fiber afferents to right-atrial injections of 5-HT were investigated in anesthetized Sprague-Dawley rats. Bolus injection of 5-HT (8 µg/kg) caused an immediate augmented breath and apnea, accompanied by hypotension and bradycardia. These initial responses were then followed by a brief pressor response and a more sustained depressor response. After a perineural treatment of both cervical vagi with capsaicin to block the conduction of C fibers, 5-HT still triggered the augmented breath, but no longer evoked the apnea, bradycardia and hypotension, indicating an involvement of C-fiber activation. The remaining augmented breath induced by 5-HT after perineural capsaicin treatment was totally eliminated by vagotomy. To further study the effect of 5-HT on CSLV afferents, activities arising from these afferents were determined using the single-fiber recording technique. Right-atrial injection of 5-HT evoked an intense discharge in CSLV afferents in a dose-dependent manner. The highest dose of 5-HT (16 µg/kg) activated 79% (19/24) of CSLV afferents which were also sensitive to capsaicin (0.8 µg/kg). The pretreatment of tropisetron, a selective antagonist of the 5-HT3 receptor, completely blocked CSLV-afferents stimulation induced by 5-HT but did not affect that by capsaicin. Furthermore, a similar afferent response of CSLV afferents was mimicked by phenylbiguanide, a selective agonist of the 5-HT3 receptor. In isolated rat lung vagal C neurons, 5-HT induced intense calcium transients in a dose-dependent manner. The highest concentration (3 µM) of 5-HT activated 67% (18/27) of the CSLV neurons. The 5-HT-induced response was totally abolished by pretreatment of tropisetron. In conclusion, 5-HT exerts an intense stimulatory effect on lung C-fiber terminals mediated through an activation of the 5-HT3 receptor, which may contribute to the airway hypersensitivity under lung inflammation.

Functional Invalidation of Putative Sudden Infant Death Syndrome-Associated Variants in the KCNH2-Encoded Kv11.1 Channel.

BACKGROUND: Heterologous functional validation studies of putative long-QT syndrome subtype 2-associated variants clarify their pathological potential and identify disease mechanism(s) for most variants studied. The purpose of this study is to clarify the pathological potential for rare nonsynonymous KCNH2 variants seemingly associated with sudden infant death syndrome. METHODS: Genetic testing of 292 sudden infant death syndrome cases identified 9 KCNH2 variants: E90K, R181Q, A190T, G294V, R791W, P967L, R1005W, R1047L, and Q1068R. Previous studies show R181Q-, P967L-, and R1047L-Kv11.1 channels function similar to wild-type Kv11.1 channels, whereas Q1068R-Kv11.1 channels accelerate inactivation gating. We studied the biochemical and biophysical properties for E90K-, G294V-, R791W-, and R1005W-Kv11.1 channels expressed in human embryonic kidney 293 cells; examined the electronic health records of patients who were genotype positive for the sudden infant death syndrome-linked KCNH2 variants; and simulated their functional impact using computational models of the human ventricular action potential. RESULTS: Western blot and voltage-clamping analyses of cells expressing E90K-, G294V-, R791W-, and R1005W-Kv11.1 channels demonstrated these variants express and generate peak Kv11.1 current levels similar to cells expressing wild-type-Kv11.1 channels, but R791W- and R1005W-Kv11.1 channels accelerated deactivation and activation gating, respectively. Electronic health records of patients with the sudden infant death syndrome-linked KCNH2 variants showed that the patients had median heart rate-corrected QT intervals <480 ms and none had been diagnosed with long-QT syndrome or experienced cardiac arrest. Simulating the impact of dysfunctional gating variants predicted that they have little impact on ventricular action potential duration. CONCLUSIONS: We conclude that these rare Kv11.1 missense variants are not long-QT syndrome subtype 2-causative variants and therefore do not represent the pathogenic substrate for sudden infant death syndrome in the variant-positive infants.

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.

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 Ca(2+), 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.

Airway extravasation induced by increasing airway temperature in ovalbumin-sensitized rats.

This study was carried out to determine whether hyperventilation of humidified warm air (HWA) induced airway extravasation in ovalbumin (Ova)-sensitized rats. Our results showed: (1) After isocapnic hyperventilation with HWA for 2 min, tracheal temperature (Ttr) was increased to 40.3°C, and the Evans blue contents in major airways and lung tissue were elevated to 651% and 707%, respectively, of that after hyperventilation with humidified room air in Ova-sensitized rats; this striking effect of HWA was absent in control rats. (2) The HWA-induced increase in Evans blue content in sensitized rats was completely prevented by a pretreatment with either L-732138, a selective antagonist of neurokinin type 1 (NK-1) receptor, or formoterol, a selective agonist of ß2 adrenoceptor. This study demonstrated that an increase in airway temperature induced protein extravasation in the major airways and lung tissue of sensitized rats, and an activation of the NK-1 receptor by tachykinins released from bronchopulmonary C-fiber nerve endings was primarily responsible.