BACKGROUND: Understanding the characteristics of pulmonary resistance and elastance in relation to the location of airway narrowing, e.g., tracheal stenosis vs. intrapulmonary airway obstruction, will help us understand lung function characteristics and mechanisms related to different airway diseases.
METHODS: In this study, we used ex vivo sheep lungs as a model to measure lung resistance and elastance across a range of transpulmonary pressures (5-30 cmH2O) and ventilation frequencies (0.125-2 Hz). We established two tracheal stenosis models by inserting plastic tubes into the tracheas, representing mild (71.8% lumen area reduction) and severe (92.1%) obstructions. For intrapulmonary airway obstruction, we induced airway narrowing by challenging the lung with acetylcholine (ACh).
RESULTS: We found a pattern change in the lung resistance and apparent lung elastance as functions of ventilation frequency that depended on the transpulmonary pressure (or lung volume). At a transpulmonary pressure of 10 cmH2O, lung resistance increased with ventilation frequency in severe tracheal stenosis, whereas in ACh-induced airway narrowing the opposite occurred. Furthermore, apparent lung elastance at 10 cmH2O decreased with increasing ventilation frequency in severe tracheal stenosis whereas in ACh-induced airway narrowing the opposite occurred. Flow-volume analysis revealed that the flow amplitude was much sensitive to ventilation frequency in tracheal stenosis than it was in ACh induced airway constriction.
CONCLUSIONS: Results from this study suggest that lung resistance and apparent elastance measured at 10 cmH2O over the frequency range of 0.125-2 Hz can differentiate tracheal stenosis vs. intrapulmonary airway narrowing in ex vivo sheep lungs.

Pulmonary surfactant serves as a barrier to respiratory epithelium but can also regulate airway smooth muscle (ASM) tone. Surfactant (SF) relaxes contracted ASM, similar to β2-agonists, anticholinergics, nitric oxide, and prostanoids. The exact mechanism of surfactant relaxation and whether surfactant relaxes hyperresponsive ASM remains unknown. Based on previous research, relaxation requires an intact epithelium and prostanoid synthesis. We sought to examine the mechanisms by which surfactant causes ASM relaxation. Organ bath measurements of isometric tension of ASM of guinea pigs in response to exogenous surfactant revealed that surfactant reduces tension of healthy and hyperresponsive tracheal tissue. The relaxant effect of surfactant was reduced if prostanoid synthesis was inhibited and/or if prostaglandin E2-related EP2 receptors were antagonized. Atomic force microscopy revealed that human ASM cells stiffen during contraction and soften during relaxation. Surfactant softened ASM cells, similarly to the known bronchodilator prostaglandin E2 (PGE2) and the cell softening was abolished when EP4 receptors for PGE2 were antagonized. Elevated levels of PGE2 were found in cultures of normal human bronchial epithelial cells exposed to pulmonary surfactant. We conclude that prostaglandin E2 and its EP2 and EP4 receptors are likely involved in the relaxant effect of pulmonary surfactant in airways.

Nicotine exposure in the context of smoking or vaping worsens airway function. Although commonly thought to exert effects through the peripheral nervous system, we previously showed airway smooth muscle (ASM) expresses nicotinic acetylcholine receptors (nAChRs), particularly alpha7 subtype (α7nAChR) with functional effects on contractility and metabolism. However, the mechanisms of nAChR regulation and downstream effects in ASM are not fully understood. Using human ASM cells from non-asthmatics vs. mild-moderate asthmatics, we tested the hypothesis that nAChR-specific ER chaperones RIC-3 and TMEM35 promote cell surface localization of α7nAChR with downstream influence on its functionality: effects exacerbated by inflammation. We found that mild-moderate asthma and exposure to pro-inflammatory cytokines relevant to asthma promote chaperone and α7nAChR expression in ASM. Downstream, ER stress was linked to nicotine/α7nAChR signaling, where RIC-3 and TMEM35 regulate nicotine-induced ER stress, Ca2+ regulation and ASM cell proliferation. Overall, our data highlights the importance α7nAChR chaperones in mediating and modulating nicotine effects in ASM towards airway contractility and remodeling.

UNASSIGNED: In the present study, the relaxant effect of crocetin on tracheal smooth muscle cells (TSM) and its possible mechanisms were evaluated.
UNASSIGNED: The study was conducted on 54 male Wistar rats in 8 groups. TSM was contracted by methacholine (10 μM) and KCl (60 mM), and the relaxant effects of four cumulative concentrations of crocetin, petal extract of saffron, and theophylline were examined on non-incubated and TSM incubated with propranolol, chlorpheniramine, diltiazem, atropine, glibenclamide, and indomethacin were investigated.
UNASSIGNED: In non-incubated TSM contracted by methacholine or KCl, crocetin and theophylline showed concentration-dependent relaxant effects (all, P<0.001). However, various concentrations of crocetin showed significantly lower relaxant effects compared to those of theophylline (all, P<0.001). In the methacholine-induced contraction of TSM, the relaxation effect of the last concentration of crocetin in the TSM incubated with propranolol was lower than in non-incubated TSM (P<0.05). In the incubated TSM with chlorpheniramine, the relaxant effects of the two last concentrations of crocetin were significantly lower than in the non-incubated tissues contracted by KCl (P<0.05 and P<0.0). The levels of EC50 crocetin in the incubated TSM with glibenclamide, chlorpheniramine, and indomethacin were markedly lower than in non-incubated (all, P<0.05).
UNASSIGNED: The results showed potent relaxation effects of crocetin on TSM and were suggested to be through stimulation of ß-adrenergic receptors, inhibition of histamine (H1) receptors, and potassium channel opening mechanisms.

The level of airway constriction in thin slices of lung tissue is highly variable. Owing to the labor-intensive nature of these experiments, determining the number of airways to be analyzed in order to allocate a reliable value of constriction in one mouse is challenging. Herein, a new automated device for physiology and image analysis was used to facilitate high throughput screening of airway constriction in lung slices. Airway constriction was first quantified in slices of lungs from male BALB/c mice with and without experimental asthma that were inflated with agarose through the trachea or trans-parenchymal injections. Random sampling simulations were then conducted to determine the number of airways required per mouse to quantify maximal constriction. The constriction of 45 ± 12 airways per mouse in 32 mice were analyzed. Mean maximal constriction was 37.4 ± 32.0%. The agarose inflating technique did not affect the methacholine response. However, the methacholine constriction was affected by experimental asthma (p = 0.003), shifting the methacholine concentration-response curve to the right, indicating a decreased sensitivity. Simulations then predicted that approximately 35, 16 and 29 airways per mouse are needed to quantify the maximal constriction mean, standard deviation and coefficient of variation, respectively; these numbers varying between mice and with experimental asthma.

A thin film of pulmonary surfactant lines the surface of the airways and alveoli, where it lowers the surface tension in the peripheral lungs, preventing collapse of the bronchioles and alveoli and reducing the work of breathing. It also possesses a barrier function for maintaining the blood-gas interface of the lungs and plays an important role in innate immunity. The surfactant film covers the epithelium lining both large and small airways, forming the first line of defense between toxic airborne particles/pathogens and the lungs. Furthermore, surfactant has been shown to relax airway smooth muscle (ASM) after exposure to ASM agonists, suggesting a more subtle function. Whether surfactant masks irritant sensory receptors or interacts with one of them is not known. The relaxant effect of surfactant on ASM is absent in bronchial tissues denuded of an epithelial layer. Blocking of prostanoid synthesis inhibits the relaxant function of surfactant, indicating that prostanoids might be involved. Another possibility for surfactant to be active, namely through ATP-dependent potassium channels and the cAMP-regulated epithelial chloride channels [cystic fibrosis transmembrane conductance regulators (CFTRs)], was tested but could not be confirmed. Hence, this review discusses the mechanisms of known and potential relaxant effects of pulmonary surfactant on ASM. This review summarizes what is known about the role of surfactant in smooth muscle physiology and explores the scientific questions and studies needed to fully understand how surfactant helps maintain the delicate balance between relaxant and constrictor needs.

G-protein-coupled receptors (GPCRs) belonging to the type 2 taste receptors (TAS2Rs) family are predominantly present in taste cells to allow the perception of bitter-tasting compounds. TAS2Rs have also been shown to be expressed in human airway smooth muscle (ASM), and TAS2R agonists relax ASM cells and bronchodilate airways despite elevating intracellular calcium. This calcium \"paradox\" (calcium mediates contraction by pro-contractile Gq-coupled GPCRs) and the mechanisms by which TAS2R agonists relax ASM remain poorly understood. To gain insight into pro-relaxant mechanisms effected by TAS2Rs, we employed an unbiased phosphoproteomic approach involving dual-mass spectrometry to determine differences in the phosphorylation of contractile-related proteins in ASM following the stimulation of cells with TAS2R agonists, histamine (an agonist of the Gq-coupled H1 histamine receptor) or isoproterenol (an agonist of the Gs-coupled β2-adrenoceptor) alone or in combination. Our study identified differential phosphorylation of proteins regulating contraction, including A-kinase anchoring protein (AKAP)2, AKAP12, and RhoA guanine nucleotide exchange factor (ARHGEF)12. Subsequent signaling analyses revealed RhoA and the T853 residue on myosin light chain phosphatase (MYPT)1 as points of mechanistic divergence between TAS2R and Gs-coupled GPCR pathways. Unlike Gs-coupled receptor signaling, which inhibits histamine-induced myosin light chain (MLC)20 phosphorylation via protein kinase A (PKA)-dependent inhibition of intracellular calcium mobilization, HSP20 and ERK1/2 activity, TAS2Rs are shown to inhibit histamine-induced pMLC20 via inhibition of RhoA activity and MYPT1 phosphorylation at the T853 residue. These findings provide insight into the TAS2R signaling in ASM by defining a distinct signaling mechanism modulating inhibition of pMLC20 to relax contracted ASM.

Crosstalk between T cells and airway smooth muscle (ASM) may play a role in modulating asthmatic airway inflammation and remodelling. Infiltrating T cells have been observed within the ASM bundles of asthmatics, and a wide range of direct and indirect interactions between T cells and ASM have been demonstrated using various in vitro and in vivo model systems. Contact-dependent mechanisms such as ligation and activation of cellular adhesion and costimulatory molecules, as well as the formation of lymphocyte-derived membrane conduits, facilitate the adhesion, bidirectional communication and transfer of materials between T and ASM cells. T cell-derived cytokines, particularly of the Th1, Th2 and Th17 subsets, modulate the secretome, proliferation and contractility of ASM cells. This review summarizes the mechanisms governing T cell-ASM crosstalk in the context of asthma. Understanding the underlying mechanistic basis is important for directing future research and developing therapeutic interventions targeted towards this complex interaction.

Succinate dehydrogenase (SDH) is a key mitochondrial enzyme involved in the tricarboxylic acid cycle, where it facilitates the oxidation of succinate to fumarate, and is coupled to the reduction of ubiquinone in the electron transport chain as Complex II. Previously, we developed a confocal-based quantitative histochemical technique to determine the maximum velocity of the SDH reaction (SDHmax) in single cells and observed that SDHmax corresponds with mitochondrial volume density. In addition, mitochondrial volume and motility varied within different compartments of human airway smooth muscle (hASM) cells. Therefore, we hypothesize that the SDH activity varies relative to the intracellular mitochondrial volume within hASM cells. Using 3D confocal imaging of labeled mitochondria and a concentric shell method for analysis, we quantified mitochondrial volume density, mitochondrial complexity index, and SDHmax relative to the distance from the nuclear membrane. The mitochondria within individual hASM cells were more filamentous in the immediate perinuclear region and were more fragmented in the distal parts of the cell. Within each shell, SDHmax also corresponded to mitochondrial volume density, where both peaked in the perinuclear region and decreased in more distal parts of the cell. Additionally, when normalized to mitochondrial volume, SDHmax was lower in the perinuclear region when compared to the distal parts of the cell. In summary, our results demonstrate that SDHmax measures differences in SDH activity within different cellular compartments. Importantly, our data indicate that mitochondria within individual cells are morphologically heterogeneous, and their distribution varies substantially within different cellular compartments, with distinct functional properties.

High serum estrogen concentrations are associated with asthma development and severity, suggesting a link between estradiol and airway hyperresponsiveness (AHR). 17β-estradiol (E2) has non-genomic effects via Ca2+ regulatory mechanisms; however, its effect on the plasma membrane Ca2+-ATPases (PMCA1 and 4) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) is unknown. Hence, in the present study, we aim to demonstrate if E2 favors AHR by increasing intracellular Ca2+ concentrations in guinea pig airway smooth muscle (ASM) through a mechanism involving Ca2+-ATPases. In guinea pig ASM, Ca2+ microfluorometry, muscle contraction, and Western blot were evaluated. Then, we performed molecular docking analysis between the estrogens and Ca2+ ATPases. In tracheal rings, E2 produced AHR to carbachol. In guinea pig myocytes, acute exposure to physiological levels of E2 modified the transient Ca2+ peak induced by caffeine to a Ca2+ plateau. The incubation with PMCA inhibitors (lanthanum and carboxyeosin, CE) partially reversed the E2-induced sustained plateau in the caffeine response. In contrast, cyclopiazonic acid (SERCA inhibitor), U-0126 (an inhibitor of ERK 1/2), and choline chloride did not modify the Ca2+ plateau produced by E2. The mitochondrial uniporter activity and the capacitative Ca2+ entry were unaffected by E2. In guinea pig ASM, Western blot analysis demonstrated PMCA1 and PMCA4 expression. The results from the docking modeling demonstrate that E2 binds to both plasma membrane ATPases. In guinea pig tracheal smooth muscle, inhibiting the PMCA with CE, induced hyperresponsiveness to carbachol. 17β-estradiol produces hyperresponsiveness by inhibiting the PMCA in the ASM and could be one of the mechanisms responsible for the increase in asthmatic crisis in women.