Airway Tone Research Articles

Airway epithelial cells promote in vitro airway smooth muscle cell proliferation by activating the Wnt/β-catenin pathway

Asthma is a common chronic inflammatory airway disease, imposing a substantial health and economic burden on society and individuals. Current treatments primarily focus on symptom relief and lung function improvement, often failing to address the underlying pathology. Thus, exploring new therapeutic approaches is crucial. Airway smooth muscle cells (ASMCs) play a key role in regulating airway tone and airflow, while abnormal ASMCs proliferation contributes to airway remodeling in asthma. Airway epithelial cells (AECs), serving as the first barrier against pathogens and allergens, also have critical immune functions.This study focuses on the interaction between AECs and ASMCs, as AECs are more accessible for drug delivery due to their location at the airway surface. Investigating this relationship could facilitate novel interventions targeting AECs to inhibit pathological ASMCs activity. In our experiment, we isolated ASMCs and AECs from healthy mice and found that AECs significantly promoted ASMCs proliferation in co-culture. RNA sequencing revealed that this process might be linked to the activation of the canonical Wnt signaling pathway in ASMCs. By using Wnt pathway inhibitors (endo-IWR1) and siRNA to disrupt Wnt receptors, we reversed this phenotype. This finding suggests that AECs may promote ASMCsproliferation by activating the Wnt pathway in ASMCs. The Wnt/β-catenin pathway appears to play an important role in ASMCs proliferation, indicating that future pathological studies should consider the potential involvement of the Wnt pathway in airway remodeling.

BMAL1 sex-specific effects in the neonatal mouse airway exposed to moderate hyperoxia.

Supplemental O2 (hyperoxia) is a critical intervention for premature infants (<34 weeks) but consequently is associated with development of bronchial airway hyperreactivity (AHR) and asthma. Clinical practice shifted toward the use of moderate hyperoxia (<60% O2), but risk for subsequent airway disease remains. In mouse models of moderate hyperoxia, neonatal mice have increased AHR with effects on airway smooth muscle (ASM), a cell type involved in airway tone, bronchodilation, and remodeling. Understanding mechanisms by which moderate O2 during the perinatal period initiates sustained airway changes is critical to drive therapeutic advancements toward treating airway diseases. We propose that cellular clock factor BMAL1 is functionally important in developing mouse airways. In adult mice, cellular clocks target pathways highly relevant to asthma pathophysiology and Bmal1 deletion increases inflammatory response, worsens lung function, and impacts survival outcomes. Our understanding of BMAL1 in the developing lung is limited, but our previous findings show functional relevance of clocks in human fetal ASM exposed to O2. Here, we characterize Bmal1 in our established mouse neonatal hyperoxia model. Our data show that Bmal1 KO deleteriously impacts the developing lung in the context of O2 and these data highlight the importance of neonatal sex in understanding airway disease.

Nicotine affects mitochondrial structure and function in human airway smooth muscle cells.

Exposure to cigarette smoke and e-cigarettes, with nicotine as the active constituent, contributes to increased health risks associated with asthma. Nicotine exerts its functional activity via nicotinic acetylcholine receptors (nAChRs), and the alpha7 subtype (α7nAChR) has recently been shown to adversely affect airway dynamics. The mechanisms of α7nAChR action in airways, particularly in the context of airway smooth muscle (ASM), a key cell type in asthma, are still under investigation. Mitochondria have garnered increasing interest for their role in regulating airway tone and adaptations to cellular stress. Here mitochondrial dynamics such as fusion versus fission, and mitochondrial Ca2+ ([Ca2+]m), play an important role in mitochondrial homeostasis. There is currently no information on effects and mechanisms by which nicotine regulates mitochondrial structure and function in ASM in the context of asthma. We hypothesized that nicotine disrupts mitochondrial morphology, fission-fusion balance, and [Ca2+]m regulation, with altered mitochondrial respiration and bioenergetics in the context of asthmatic ASM. Using human ASM (hASM) cells from nonasthmatics, asthmatics, and smokers, we examined the effects of nicotine on mitochondrial dynamics and [Ca2+]m. Fluorescence [Ca2+]m imaging of hASM cells with rhod-2 showed robust responses to 10 μM nicotine, particularly in asthmatics and smokers. In both asthmatics and smokers, nicotine increased the expression of fission proteins while decreasing fusion proteins. Seahorse analysis showed blunted oxidative phosphorylation parameters in response to nicotine in these groups. α7nAChR siRNA blunted nicotine effects, rescuing [Ca2+]m, changes in mitochondrial structural proteins, and mitochondrial dysfunction. These data highlight mitochondria as a target of nicotine effects on ASM, where mitochondrial disruption and impaired buffering could permit downstream effects of nicotine in the context of asthma.NEW & NOTEWORTHY Asthma is a major healthcare burden, which is further exacerbated by smoking. Recognizing the smoking risk of asthma, understanding the effects of nicotine on asthmatic airways becomes critical. Surprisingly, the mechanisms of nicotine action, even in normal and especially asthmatic airways, are understudied. Accordingly, the goal of this research is to investigate how nicotine influences asthmatic airways in terms of mitochondrial structure and function, via the a7nAChR.

The Role of Long-Acting Antimuscarinic Agents in the Treatment of Asthma.

The journey of using anticholinergics in the treatment of asthma started with anticholinergic-containing plants such as Datura stramonium and Atropa belladonna, followed by ipratropium bromide and continued with tiotropium, glycopyrronium, and umeclidinium. Although antimuscarinics were used in the maintenance treatment of asthma over a century ago, after a long time (since 2014), it has been recommended to be used as an add-on long-acting antimuscarinic agent (LAMA) therapy in the maintenance treatment of asthma. The airway tone controlled by the vagus nerve is increased in asthma. Allergens, toxins, or viruses cause airway inflammation and inflammation-related epithelial damage, increased sensory nerve stimulation, ganglionic and postganglionic acetylcholine (ACh) release by inflammatory mediators, intensification of ACh signaling at M1 and M3 muscarinic ACh receptors (mAChRs), and dysfunction of M2 mAChR. Optimal anticholinergic drug for asthma should effectively block M3 and M1 receptors, but have minimal effect on M2 receptors. Tiotropium, umeclidinium, and glycopyrronium are anticholinergic agents with this feature. Tiotropium has been used in a separate inhaler as an add-on treatment to inhaled corticosteroid (ICS)/long-acting β2-agonist (LABA), and glycopyrronium and umeclidinium have been used in a single inhaler as a combination of ICS/LABA/LAMA in asthma in recent years. Guidelines recommend this regimen as an optimization step for patients with severe asthma before initiating any biologic or systemic corticosteroid therapy. In this review, the history of antimuscarinic agents, their effectiveness and safety in line with randomized controlled trials, and real-life studies in asthma treatment will be discussed according to the current data.

Influence of obesity on the tone of bronchial smooth muscles in rats

Background. Overweight and obesity are key factors for the occurrence of many morphofunctional disorders in organs and tissues, including bronchopulmonary system.The aim. To study the influence of metabolic disorders that occur against the background of obesity on the state of the airways tone in rats.Materials and methods. Obesity in male Wistar rats was induced using a high-fat and high-carbohydrate diet. In animals, body weight and fat mass were measured, and the heart-lung complex was extracted. In blood serum, the levels of glucose, insulin, leptin, triglycerides, and cholesterol were assessed. Bronchoalveolar lavage fluid was obtained by an open method, in which the concentration of protein, interleukin (IL) 6 and IL-10 was determined. The contractile activity of the isolated bronchial smooth muscle segments was studied using mechanographic method. The effect of acetylcholine (10–7–10–4 M), indomethacin (10–5 M), and forskolin (10–7–10–5 M) on the changes in the tone of airway smooth muscles was assessed.Results. High-fat and high-carbohydrate diet caused an increase in body weight, visceral obesity, hyperglycemia, insulin resistance, leptinemia, dyslipidemia in rats of the experimental group. In the bronchoalveolar lavage fluid of experimental animals, an increase in the content of protein and IL-6 was found, which positively correlated with the level of leptin and the fat mass. In obese rats, the contractile responses of bronchial smooth muscle segments increased in response to the effect of the cholinergic agent acetylcholine. The bronchoconstrictor effect of acetylcholine was reduced by the cyclooxygenase inhibitor indomethacin. In turn, the adenylate cyclase activator forskolin caused relaxation of the airway segments smooth muscles in rats of both groups, which was more pronounced in the experimental group.Conclusion. The obtained results indicate that the change in the reactivity of the respiratory tract can be the cause of bronchospastic conditions in obesity and of the inflammatory reaction in the respiratory system induced by obesity.

Muscarinic modulation increases excitability inspiratory and non-inspiratory hypoglossal motoneurons in mice postnatal days 0-5

Hypoglossal motoneurons (XII MNs) innervate the tongue and their activity normally contributes to stiffening of the airway to maintain airway patency. During sleep, there is normal reduction in airway tone. The specific mechanisms that contribute to decreased airway tone during sleep remain to be completely resolved, although research from adult rodents suggests activation of muscarinic acetylcholine receptors (mAChRs) is an important contributor by inhibiting XII MNs. Conversely, data from neonatal rodents, have shown that muscarinic modulation leads to an overall excitatory effect in XII MNs. Therefore, the overarching objective of this research project is to uncover the mechanisms through which muscarinic modulation can influence the excitability of XII MNs across postnatal maturation. Rhythmic medullary slices from neonatal mice (postnatal day 0-5) were used for whole cell electrophysiology experiments to test the effects of activating mAChRs at XII MNs in both non-inspiratory and inspiratory XII MNs. Data are reported as mean±SD. We hypothesized that mAChR activation at XII MNs would increase excitability of non-inspiratory and inspiratory XII MNs, and potentiate inspiratory bursting in inspiratory XII MNs. Starting at -60 mV, muscarinic modulation depolarized non-inspiratory and inspiratory XII MNs similarly (3.6±2.6 vs 4.6±2.1 mV, n=12, p&gt;0.05), and had no effect on input resistance (CTL vs Musc; non-insp: 120±23 vs 128±33 MΩ; inspiratory: 140±28 vs 137±32 MΩ; n =12; p&gt;0.05). Muscarinic modulation can increase the hyperpolarization-activated cation current (I h ), but we observed no difference in the sag potential in response to -150 (CTL vs Musc; non-insp: 2.3±2.3 vs 3.8±3.0 mV; inspiratory: 2.7±2.8 vs 4.9±2.8 mV; n =12; p &gt; 0.05), -300 (CTL vs Musc; non-insp: 9.0±7.8 vs 11.7±8.0 mV; inspiratory: 9.9±6.4 vs 12.9±7.5 mV; n=12; p&gt;0.05), and -500 pA (CTL vs Musc; non-insp: 21.9±15.0 vs 23.6±14.2 mV; inspiratory: 22.0±9.5 vs 23.5±9.5 mV; n=12; p&gt;0.05) current steps from -45 mV in non-inspiratory compared to inspiratory XII MNs. Muscarinic modulation significantly increased the firing frequency after muscarine application for inspiratory cells with the current step injections 100 pA (CTL vs Musc; 14.7±6.8 vs 21.1±6.4 Hz, n=9 and 10; p&lt;0.05) and 175 pA (CTL vs Musc; 19.5±6.4 vs 25.0±9.4 Hz, n=11 and 8; p&lt;0.05). However, muscarinic modulation did not affect the slope of firing rate/injected current relationship for non-inspiratory and inspiratory XII MNs (CTL vs Musc; non-insp: 0.16±0.12 vs 0.15±0.04 Hz/pA, n=9; inspiratory: 0.12±0.02 vs 0.16±0.07 Hz/pA, n=11 and 12; p&gt;0.05). Finally, muscarinic modulation significantly increased inspiratory burst amplitude (CTL vs Musc; 15.2±3.1 vs 18.3±4.5 mV; n=3; p&lt;0.05), and a trend to increase inspiratory burst area (CTL vs Musc; 4260±779 vs 5354±3063 mV ms, n=3; p=0.08). These data indicated that muscarinic modulation has similar effects on non-inspiratory and inspiratory XII MNs early in postnatal growth. Funding: NIH This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Centrally derived gasotransmitter modulation of hypoglossal motoneuron activity

Airway obstruction during inspiration is a hallmark trait of sleep apnea (SA). In mice, genetic elimination of the CO producing enzyme, heme oxygenase-2 (HO-2), leads to a SA phenotype predominately associated with recurrent airway obstruction. This phenotype is mitigated by either inhibition or genetic ablation of the H2S producing enzyme, cystathionine g-lysase (CSE). While these observations indicate that mutual interactions between HO-2/CO and CSE/H2S are important for regulating airway clearance, the extent of contribution from these interaction in the central nervous system to airway tone are unclear. Here, we determine how HO-2/CO and CSE/H2S impact inspiratory output from the hypoglossal nucleus (XIIn) in the isolated rhythmic brainstem slice preparation. We hypothesize that these enzymes and their respective gasotransmitter products locally interact in the inspiratory circuit to modulate the input-output (I/O) relationship between preBötC and XIIn. Electrophysiological studies conducted in brainstem slices from mice (P5-12) showed that HO-2 dysregulation, using either in wildtype slices exposed to Cr (III) Mesoporphyrin IX chloride or in slices from HO-2 null mice, increased transmission failure from preBötC to XIIn and altered the I/O relationship between the rhythm generating network and XIIn. HO-2 dysregulation also increased subnetwork activity in the preBötC that was associated with a decrease in amplitude regularity. While many transmission failures to the XIIn were associated with preBötC subnetwork activity, simultaneous recordings from the preBötC, premotor area, and the XIIn revealed that preBötC subnetwork activity was reliably transmitted to the premotor field but not to the XIIn. Dual extracellular and intracellular recordings from the preBötC and individual XIIn neurons under disinhibited conditions revealed that HO-2 dysregulation reduced inspiratory drive currents received by XIIn neurons, decreased the intrinsic excitability of XIIn neurons while also reducing the amount of preBötC subnetwork activity. Additionally, targeted inhibition of CSE/H2S activity using L-PAG or by genetic elimination of CSE in HO-2 null mice mitigated these effects of HO-2 dysregulation. Thus, the mutual interactions HO-2/CSE and CSE/H2S in the medullary brainstem appear to modulate XIIn output by: (1) causing a E/I imbalance that promotes preBötC subnetwork activity; (2) suppressing inspiratory drive received in XIIn; and (3) suppressing intrinsic excitability of XIIn neurons. These findings may be key to understanding the action by which central gasotransmitters actions contribute to upper airway muscle regulation. This work was supported by NIH: P01-HL-44454; R01NS107421. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neuronal muscarinic acetylcholine receptor subtype modulation of inspiratory bursting at hypoglossal motoneurons in developing neonatal mice in vitro

Muscarinic acetylcholine receptor modulation contributes to changes in hypoglossal motoneuron excitability through pre- and post-synaptic effects, an important mechanism for regulation of airway tone during sleep. Previous data indicate a net excitatory effect of muscarinic modulation of inspiratory bursting in neonatal mouse brainstem slices, yet a net inhibitory effect on inspiratory bursting in adult rats. We tested the hypothesis that a developmental shift in muscarinic modulation impacts hypoglossal motoneurons, using neuroanatomical and electrophysiological methods. Immunofluorescence confirmed hypoglossal motoneurons express M1, M2, M3, and M5 muscarinic subtypes. We observed maturational decreases in M1 and M5 labeling intensity, M3 labeling intensity generally decreased with postnatal (P) age, although there was a transient increase in expression at P17. M2 labeling intensity did not change, although distribution of M2 labeling with postnatal maturation may change. Next, using the in-vitro rhythmic slice preparation from CD1 mice of either sex, local application of muscarine (100 μM) into the hypoglossal nucleus was tested in mice P0-14. The net effect of applying muscarine results in excitatory output after local application into the rhythmic slice because each data point is a positive percent change from control. The magnitude of this potentiation appears to lessen as the mice age toward P14. To evaluate the mechanisms through which the magnitude of muscarinic potentiation of inspiratory bursting changes with postnatal maturation, we next assessed the role of excitatory and inhibitory muscarinic receptor subtypes. Blocking M1 locally (pirenzepine, 100 μM) significantly decreased muscarine-mediated potentiation of inspiratory bursting (to 72±14% of control muscarine response, n=9, P0-5). Preliminary data indicate bath application of 4-DAMP (M3 antagonist) attenuated muscarinic potentiation of inspiratory bursting (range: to 7%-25% of control burst response, n=3, P0-5). Modulating M5 locally with a positive allosteric modulator VU 0238429 (1000 μM) had little effect on burst amplitude (119±4% of control burst response, n=3, P0-6). Blocking M2 locally (methoctramine, 2μM) showed no effect on inspiratory burst potentiation after applying muscarine (181±65% vs 187±69%, n=7, P0-5). Bath application of methoctramine did not have a significant effect on inspiratory burst potentiation by muscarine (179±65% vs control 182±90%, n=10) in P0-5 mice while preliminary analysis suggests an increasing effect of blocking M2 with postnatal maturation (147±56% vs control 134±30%, n=4, P9-14). These data support excitatory muscarinic receptor subtypes contributing to the excitatory response early in postnatal maturation. Based on our neuroanatomical data, we speculate the increasing inhibitory response to muscarinic modulation with postnatal maturation is due to a decreased contribution of excitatory muscarinic receptor subtypes. NIH R15HL148870 (Revill) and NIH/NHLBI R25 HL126140 (Moreno, Garcia, Parthasarathy) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Methodical control of the difficult pediatric airway: two case reports

BackgroundManagement of children who present with a history of impossible mask ventilation or difficult tracheal intubation is fraught with challenges. Despite this, the “airway stress test” of an inhalational induction is frequently employed risking airway obstruction, breath holding, apnea, and laryngospasm.Case presentationsWe present two cases of children with anticipated difficult airway management. The first child (14-year-old African American boy) had severe mucopolysaccharidosis with a history of failed anesthetic induction and failed airway management. The second child (3-year-old African American girl) had progressive lymphatic infiltration of the tongue, resulting in severe macroglossia. We describe a technique that forgoes inhalational induction, incorporates recent pediatric airway guidelines, and provides a greater margin of safety. The technique encompasses the use of drugs that facilitate sedation for intravenous access, without respiratory depression or airway obstruction, titrated use of medications to achieve anesthetic depth while preserving ventilatory drive and airway tone, and the continuous provision of directed oxygen flow during airway manipulation. Propofol and volatile gases were avoided to preserve airway tone and respiratory drive.ConclusionsWe emphasize that an intravenous induction technique utilizing medications that preserve airway tone and ventilatory drive, and the use of continuous oxygen flow throughout airway manipulation, allows for successful management of children with a difficult airway. The common practice of volatile inhalational induction should be avoided in anticipated difficult pediatric airways.

The endocannabinoid anandamide is an airway relaxant in health and disease

Chronic obstructive airway diseases are a global medical burden that is expected to increase in the near future. However, the underlying mechanistic processes are poorly understood so far. Herein, we show that the endocannabinoid anandamide (AEA) induces prominent airway relaxation in vitro and in vivo. In contrast to 2-arachidonlyglycerol-induced airway relaxation, this is mediated by fatty acid amide hydrolase (FAAH)-dependent metabolites. In particular, we identify mouse and also human epithelial and airway smooth muscle cells as source of AEA-induced prostaglandin E2 production and cAMP as direct mediator of AEA-dependent airway relaxation. Mass spectrometry experiments demonstrate reduced levels of endocannabinoid-like compounds in lungs of ovalbumin-sensitized mice indicating a pathophysiological relevance of endocannabinoid signalling in obstructive airway disease. Importantly, AEA inhalation protects against airway hyper-reactivity after ovalbumin sensitization. Thus, this work highlights the AEA/FAAH axis as a critical regulator of airway tone that could provide therapeutic targets for airway relaxation.

Tragus pressure-guided removal of airway devices for safe emergence from sedation: A randomised controlled trial

Dear Editor, Emergence from anaesthesia and deep sedation is the transition from unconsciousness to the return of awareness and airway reflexes. The chief patterns of unsafe recovery include sudden unpredictable emergence or delayed return of airway tone and reflex with risk of airway obstruction.1 Agitation includes straining, sitting up, screaming or shouting. Even with supraglottic

Effects of the Lubo cervical collar on airway patency in awake adults – A magnetic resonance imaging study

Introduction: Intended for use by prehospital first responders, the Lubo TM cervical collar is an adjustable, radiolucent, single-use device that incorporates a mechanical jaw thrust mechanism. The combination enables non-invasive airway management in cases of trauma where cervical motion restriction is necessary. The potential benefits include use as an airway adjuvant maintaining upper airway patency, reducing provider task loading. The limited research on the device efficacy and safety requires further investigation.Methods: A randomized, crossover, interventional study was performed to compare mean differences in airway patency at the level of the uvula, epiglottis, tongue and soft palate with and without the Lubo collar in awake volunteers using magnetic resonance imaging (MRI). Fourteen participants each underwent two MRI scans of the upper airway: A control scan with no Lubo collar, and an intervention scan with the Lubo collar applied and jaw thrust mechanism activated. Two independent radiologists measured anterior-posterior diameter of the airway at four anatomical levels on the resulting MRI images.Results: There was no significant difference in mean airway diameter between the control and intervention measurements at any level. Mean (SD; 95% CI: p-value) differences were 0.9 mm (-2.38; 2.3 to 0.5; p=0.17) at the epiglottis, 0.5 mm (1.6; -0.5 to 1.4; p=0.29) at the soft palate, 0.2 mm (2.86; -1.4 to 1.9; p = 0.78) at the tongue, 0.4 mm (4.04; -1.9 to 2.7; p = 0.72) at the uvula.Conclusion: The Lubo TM airway collar did not show a significant change in upper airway patency at four anatomical levels measured in awake adult participants. Further research is required to investigate its clinical use in patients that are unable to maintain upper airway tone. Groups of interest would include trauma, obstructive sleep apnoea, obesity and patients under general anaesthesia.

The differential roles of the two NO-GC isoforms in adjusting airway reactivity.

The enzyme, nitric oxide-sensitive guanylyl cyclase (NO-GC), is activated by binding NO to its prosthetic heme group and catalyzes the formation of cGMP. The NO-GC is primarily known to mediate vascular smooth muscle relaxation in the lung, and inhaled NO has been successfully used as a selective pulmonary vasodilator. In comparison, NO-GC's impact on the regulation of airway tone is less acknowledged and, most importantly, little is known about the issue that NO-GC signaling is accomplished by two isoforms: NO-GC1 and NO-GC2, implying the existence of distinct "cGMP pools." Herein, we investigated the functional role of the NO-GC isoforms in respiration by measuring lung function parameters of isoform-specific knockout (KO) mice using noninvasive and invasive techniques. Our data revealed the participation and ongoing influence of NO-GC1-derived cGMP in the regulation of airway tone by showing that respiratory resistance was enhanced in NO-GC1-KOs and increased more pronouncedly after the challenge with the bronchoconstrictor methacholine. The tissue resistance and stiffness of NO-GC1-KOs were also higher because of narrowed airways that cause tissue distortion. Contrariwise, NO-GC2-KOs displayed reduced tissue elasticity, elastic recoil, and airway reactivity to methacholine, which did not even increase in an ovalbumin model of asthma that induced hyperresponsiveness in NO-GC1-KOs. In addition, conscious NO-GC2-KOs showed a higher breathing rate with a shorter duration of inspiration and expiration time, which remained faster even in the presence of bronchoconstrictors that slow down breathing. Thus, we provide evidence of two distinct NO/cGMP pathways in airways, accomplished by either NO-GC1 or NO-GC2, adjusting differentially the airway reactivity.

Platelet-derived growth factor (PDGF)-BB regulates the airway tone via activation of MAP2K, thromboxane, actin polymerisation and Ca2+-sensitisation

BackgroundPDGFR-inhibition by the tyrosine kinase inhibitor (TKI) nintedanib attenuates the progress of idiopathic pulmonary fibrosis (IPF). However, the effects of PDGF-BB on the airway tone are almost unknown. We studied this issue and the mechanisms beyond, using isolated perfused lungs (IPL) of guinea pigs (GPs) and precision-cut lung slices (PCLS) of GPs and humans.MethodsIPL: PDGF-BB was perfused after or without pre-treatment with the TKI imatinib (perfused/nebulised) and its effects on the tidal volume (TV), the dynamic compliance (Cdyn) and the resistance were studied. PCLS (GP): The bronchoconstrictive effects of PDGF-BB and the mechanisms beyond were evaluated. PCLS (human): The bronchoconstrictive effects of PDGF-BB and the bronchorelaxant effects of imatinib were studied. All changes of the airway tone were measured by videomicroscopy and indicated as changes of the initial airway area.ResultsPCLS (GP/human): PDGF-BB lead to a contraction of airways. IPL: PDGF-BB decreased TV and Cdyn, whereas the resistance did not increase significantly. In both models, inhibition of PDGFR-(β) (imatinib/SU6668) prevented the bronchoconstrictive effect of PDGF-BB. The mechanisms beyond PDGF-BB-induced bronchoconstriction include activation of MAP2K and TP-receptors, actin polymerisation and Ca2+-sensitisation, whereas the increase of Ca2+ itself and the activation of EP1–4-receptors were not of relevance. In addition, imatinib relaxed pre-constricted human airways.ConclusionsPDGFR regulates the airway tone. In PCLS from GPs, this regulatory mechanism depends on the β-subunit. Hence, PDGFR-inhibition may not only represent a target to improve chronic airway disease such as IPF, but may also provide acute bronchodilation in asthma. Since asthma therapy uses topical application. This is even more relevant, as nebulisation of imatinib also appears to be effective.

0820 Sleep and Oculopharyngeal Muscular Dystrophy: disease progression affecting ventilatory needs and treatment of sleep-disordered breathing

Abstract Introduction Oculopharyngeal muscular dystrophy (OPMD) is an autosomal-dominant, late-onset, and progressive disease characterized by ptosis and dysphagia, sometimes proximal limb weakness and gait abnormalities. It often presents in patients in their 50s. The progressive functional decline of the pharyngeal muscles results in feeding difficulties and aspiration; however, patients may also have risk of nocturnal hypoventilation and sleep apnea, complicated by variable airway obstruction and compliance. Report of Cases: Using retrospective chart review, we identified patients with a known diagnosis of OPMD treated at the Raymond G. Murphy VA Sleep Center. We present a case where OPMD progression necessitated increased ventilatory support and affected positive airway pressure (PAP) compliance. A 58-year-old male with OPMD, DMT2, depression, and memory impairment underwent home sleep apnea testing showing severe OSA (REI 32.7, SpO2 nadir 72%). He started Auto-PAP 6-16 cwp and presented to discuss issues tolerating PAP. Pressures were lowered, but he continued to require maximum pressures without increased utilization. An in-lab CPAP titration showed treatment-emergent centrals but did not find optimal pressures due to limited sleep time. Having failed CPAP, he returned for an ASV titration which controlled his apnea in lateral position. Patient was switched to auto ASV to increase efficacy and comfort. Two months later he discontinued ASV due to frustration with disease progression and feeling unable to breathe deeply with the machine. Nocturnal oxygen at 1L was ordered while he awaited Neurology consult for OPMD. Later, concerned about progressive dyspnea, he resumed ASV, now with 3L O2 bleed. Given suspicion of hypoventilation (bicarbs 27-30), and that ASV could not adjust to his continually changing airway tone with his OPMD, he was switched to iVAPS. This resulted in good control of his sleep apnea, tidal volumes and minute ventilation. However, he reported pressures felt too high, returned to ASV for a period, then discontinued PAP altogether. Conclusion Patients with OPMD and sleep apnea require close follow-up as their disease progression may affect their ventilatory support needs. These patients may require more complex PAP modalities, such as AVAPs, and routine PFTs to help determine timing of ENT involvement for surgical airway planning. Support (If Any)

Gaseotransmitters Modulate Inspiratory Drive from the Hypoglossal Nucleus

Gaseotransmitters, carbon monoxide (CO) and hydrogen sulfide (H2S), play an important role in maintaining proper function of cardio‐respiratory systems. Previous work has demonstrated that Heme Oxygenase‐2 knock out mice (HO‐2‐/‐) develop a sleep apnea phenotype characterized predominantly by obstructive apneas, which could be normalized by either treating HO‐2‐/‐ with L‐propargylglycine (L‐PAG) an inhibitor of cystathionine gamma‐lysase (CSE) and H2S synthesizing enzyme or in HO‐2+ CSE double null mice. Obstructive apneas are often caused by a loss in upper airway tone, we sought to examine how H2S and CO signaling impacts inspiratory activity from the preBötC and the hypoglossal nucleus (XIIn). We hypothesized that HO‐2 and CSE are involved in the regulation of inspiratory motor output from XIIn. Immunohistochemical analysis revealed many ChAT+ cells of XIIn are positive for HO‐2 and CSE. Biochemical analysis showed elevated H2S abundance in XIIn of HO‐2‐/‐mice. Electrophysiological recordings demonstrated transmission failure from preBötC to XIIn in response to NaHS, a H2S donor or Cr (III) Mesoporphyrin IX chloride (ChrMP459), an inhibitor of HO in brain slices from wild type mice. Furthermore, ChrMP459 reduced inspiratory drive currents received by XIIn neurons and suppressed excitability in XIIn neurons. Brain slices from HO‐2‐/‐mice showed: (1) a larger rate of transmission failure between preBötC and XIIn motor pools; and (2) smaller drive currents from individual XIIn neurons. Brain slices from HO‐2+ CSE double null mice displayed reduced transmission failures from preBötC to XIIn. Furthermore, L‐PAG mitigated transmission failures and increased the magnitude of inspiratory drive currents in XIIn neurons in HO‐2‐/‐mice. These findings demonstrate that HO‐2 and CSE regulate inspiratory drive from XIIn and suggest potential role for gaseotransmitters in regulating upper airway tone.

Sedation and analgesia from prolonged pain and stress during mechanical ventilation in preterm infants: is dexmedetomidine an alternative to current practice?

Mechanical ventilation is an uncomfortable and potentially painful intervention. Opioids, such as morphine and fentanyl, are used for analgesia and sedation but there is uncertainty whether they reduce pain in mechanically ventilated infants. Moreover, there may be short-term and long-term adverse consequences such as respiratory depression leading to prolonged mechanical ventilation and detrimental long-term neurodevelopmental effects. Despite this, opioids are widely used, possibly due to a lack of alternatives.Dexmedetomidine, a highly selective alpha-2-adrenergic agonist with analgesic and sedative effects, currently approved for adults, has come into use in newborn infants. It provides analgesia and simulates natural sleep with maintenance of spontaneous breathing and upper airway tone. Although data on pharmacokinetics–pharmacodynamics in preterm infants are scant, observational studies report that using dexmedetomidine in conjunction with opioids/benzodiazepines or on its own can reduce the cumulative exposure to opioids/benzodiazepines. As it does not cause respiratory depression, dexmedetomidine could enable quicker weaning and extubation. Dexmedetomidine has also been suggested as an adjunct to therapeutic hypothermia in hypoxic ischaemic encephalopathy and others have used it during painful procedures and surgery. Dexmedetomidine infusion can cause bradycardia and hypotension although most report clinically insignificant effects.The increasing number of publications of observational studies and clinical use demonstrates that dexmedetomidine is being used in newborn infants but data on safety and efficacy are scant and not of high quality. Importantly, there are no data on long-term neurodevelopmental impact on preterm or term-born infants. The acceptance of dexmedetomidine in routine clinical practice must be preceded by clinical evidence. We need adequately powered and well-designed randomised controlled trials investigating whether dexmedetomidine alone or with opioids/benzodiazepines in infants on mechanical ventilation reduces the need for opioids/benzodiazepine and improves neurodevelopment at 24 months and later as compared with the use of opioids/benzodiazepines alone.

Role of airway smooth muscle cell phenotypes in airway tone and obstruction in guinea pig asthma model

BackgroundAirway obstruction (AO) in asthma is driven by airway smooth muscle (ASM) contraction. AO can be induced extrinsically by direct stimulation of ASM with contractile agonists as histamine, or by indirect provocation with antigens as ovalbumin, while the airway tone is dependent on intrinsic mechanisms. The association of the ASM phenotypes involved in different types of AO and airway tone in guinea pigs was evaluated.MethodsGuinea pigs were sensitized to ovalbumin and challenged with antigen. In each challenge, the maximum OA response to ovalbumin was determined, and before the challenges, the tone of the airways. At third challenge, airway responsiveness (AR) to histamine was evaluated and ASM cells from trachea were disaggregated to determinate: (a) by flow cytometry, the percentage of cells that express transforming growth factor-β1 (TGF-β1), interleukin-13 (IL-13) and sarco-endoplasmic Ca2+ ATPase-2b (SERCA2b), (b) by RT-PCR, the SERCA2B gene expression, (c) by ELISA, reduced glutathione (GSH) and, (d) Ca2+ sarcoplasmic reticulum refilling rate by microfluorometry. Control guinea pig group received saline instead ovalbumin.ResultsAntigenic challenges in sensitized guinea pigs induced indirect AO, AR to histamine and increment in airway tone at third challenge. No relationship was observed between AO induced by antigen and AR to histamine with changes in airway tone. The extent of antigen-induced AO was associated with both, TGF-β1 expression in ASM and AR degree. The magnitude of AR and antigen-induced AO showed an inverse correlation with GSH levels in ASM. The airway tone showed an inverse association with SERCA2b expression.ConclusionsOur data suggest that each type of AO and airway tone depends on different ASM phenotypes: direct and indirect AO seems to be sensitive to the level of oxidative stress; indirect obstruction induced by antigen appears to be influenced by the expression of TGF-β1 and the SERCA2b expression level plays a role in the airway tone.

Computational characterization of transducer recognition of β2 adrenergic receptor

As an important drug target, β2 adrenergic receptor (B2AR) regulates many physiological processes, including cardiac function, airway tone and metabolic functions. The selective coupling between B2AR and specific transducers is critical for the physiological action of the receptor. However, the molecular mechanism by which B2AR recognizes different transducers remains elusive. Here, molecular dynamics simulations of B2AR binding to three functionally important transducers (Gs, Gi and β-arrestin 1) unveiled distinct binding modes of the receptor. Involving transmembrane helices TMs 2–7 and intracellular loops ICLs 2–3, different binding interfaces for Gs and β-arrestin 1 were identified in the simulation models and further validated by various assays. The distinct recognition mode of B2AR for Gi was computationally characterized. Insights into receptor-transducer communication not only enhance our understanding of signaling bias, but also offer hints for rational drug design targeting specific signaling pathways of G-protein coupled receptors (GPCRs).

Comparative Genomic and Network Analysis of nNOS by Using Different Bioinformatics Approaches

Introduction: Nitric Oxide (NO) is a diatomic free radical gaseous molecule that is formed from L-arginine through NOS (Nitric oxide synthase) catalyzed reaction. NO controls vascular tone (hence blood pressure), insulin secretion, airway tone, and peristalsis, and is involved in angiogenesis (growth of new blood vessels) and development of the nervous system. In the CNS, NO is an important messenger molecule, which is involved in various major functions in the brain. NOS has been classified into three isoforms, including nNOS (neuronal NOS), eNOS (endothelial NOS) and iNOS (inducible NOS). NOS1 is localized on chromosome 12 consisting of 1434 amino acids and 161 KDa molecular weight. nNOS is involved in synaptic transmission, regulating the tone of smooth muscles and penile erection. We studied NOS1 gene and protein network analysis through in silico techniques as human nNOS sequence was fetched from GenBank and its homologous sequences were retrieved through BLAST search. Moreover, the results of this study exploit the role of NOS1 in various pathways, which provide ways to regulate it in various neurodegenerative diseases. Background: Previous research has revealed the role of Nitric Oxide (NO) formed from L-arginine through NOS (Nitric Oxide Synthase) as physiological inter/intra-cellular messenger in central as well as peripheral nervous systems. The diverse functions of NOS include insulin secretion, airway tone, vascular tone regulation, and in brain, it is involved in differentiation, development, synaptic plasticity and neurosecretion. Objective: The objective of this study is to unravel the role of neuronal Nitric Oxide Synthase (nNOS) in different pathways and its involvement as therapeutic target in various neurodegenerative disorders that can surely provide ways to regulate its activity in different aspects. Materials and Methods: In this study, we employed various bioinformatics tools and databases initiating the study by fetching the neuronal Nitric Oxide Synthase (nNOS) sequence (GenBank) to find its homologous sequences(BLAST) and then exploring its physical properties and post translational modifications, enhancing the research by network analysis (STRING), leading to its functional enrichment (Panther). Results: The results positively support the hypothesis of its role in various pathways related to neurodegeneration and its interacting partners are the probable therapeutic targets of various neurodegenerative diseases focusing on specifically multi-target analysis. Conclusion: This study considered evolutionary trend of physical, chemical and biological properties of NOS1 through different phyla. The neuronal Nitric Oxide Synthase (nNOS), being one of the three isoforms of NOS (Nitric Oxide Synthase), is found to be involved in more pathways than just forming Nitric Oxide. This research provides the base for further neurological research.