Regulation Of Airway Tone Research Articles

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.

Regulation of Myosin Light-Chain Phosphatase Activity to Generate Airway Smooth Muscle Hypercontractility.

Smooth muscle is a central structure involved in the regulation of airway tone. In addition, it plays an important role in the development of some pathologies generated by alterations in contraction, such as hypercontractility and the airway hyperresponsiveness observed in asthma. The molecular processes associated with smooth muscle contraction are centered around myosin light chain (MLC) phosphorylation, which is controlled by a balance in the activity of myosin light-chain kinase (MLCK) and myosin light-chain phosphatase (MLCP). MLCK activation depends on increasing concentrations of intracellular Ca2+, while MLCP activation is independent of Ca2+. MLCP contains a phosphatase subunit (PP1c) that is regulated through myosin phosphatase target subunit 1 (MYPT1) and other subunits, such as glycogen-associated regulatory subunit and myosin-binding subunit 85 kDa. Interestingly, MLCP inhibition may contribute to exacerbation of smooth muscle contraction by increasing MLC phosphorylation to induce hypercontractility. Many pathways inhibiting MLCP activity in airway smooth muscle have been proposed and are focused on inhibition of PP1c, inhibitory phosphorylation of MYPT1 and dissociation of the PP1c-MYPT1 complex.

Lipid Mediator Quantification in Isolated Human and Guinea Pig Airways: An Expanded Approach for Respiratory Research.

The clinical importance of prostaglandins and leukotrienes in asthma is well recognized; however, the biochemical role of other lipid mediators (often termed oxylipins) in the regulation of airway tone and inflammation remains unclear. We therefore developed a workflow to investigate oxylipin physiology and pharmacology in two in vitro models, the intact human bronchus and the guinea pig trachea. Airways were isolated and smooth muscle contraction was measured in an organ bath following stimulation with either anti-IgE or ovalbumin. The associated release of oxylipins over time into the organ bath was quantified using three developed LC-MS/MS methods capable of collectively measuring 130 compounds. Oxylipin extraction recoveries were 71% on average, method accuracy was 90-98%, coefficient of variation was 4.3-9.4%, and matrix effects were on average 11%. At baseline, low levels of primarily prostaglandins and associated metabolites were observed in both tissue preparations. The mast cell-induced airway constriction caused release of leukotrienes and further elevations in prostaglandin levels. In total, 57 oxylipins from the human bronchus, and 42 from guinea pig trachea, were detected at 60 min post-stimulation in the organ bath. Chiral analysis demonstrated that 5-hydroxyeicosatetraenoic acid (5-HETE) in the human bronchus preparation was not produced by 5-LOX enzymatic activity (enantiomeric excess [ee] = 10%), as opposed to 12( S)-HETE, 14( S)-, and 17( S)-hydroxy docosahexaenoic acid (HDoHE; ee = 100%), highlighting that chiral chromatography is necessary for correct biological interpretation. Unexpectedly, prostaglandin D2 and its metabolites remained elevated 24 h after the challenges, suggesting a sustained activation of mast cells not previously described. The reported translational methodology provides a new platform for comprehensive studies to elucidate the origin and functions of individual oxylipins in various airway responses.

Caveolin-1: Functional Insights into Its Role in Muscarine- and Serotonin-Induced Smooth Muscle Constriction in Murine Airways.

An increased bronchoconstrictor response is a hallmark in the progression of obstructive airway diseases. Acetylcholine and 5-hydroxytryptamine (5-HT, serotonin) are the major bronchoconstrictors. There is evidence that both cholinergic and serotonergic signaling in airway smooth muscle (ASM) involve caveolae. We hypothesized that caveolin-1 (cav-1), a structural protein of caveolae, plays an important regulatory role in ASM contraction. We analyzed airway contraction in different tracheal segments and extra- and intrapulmonary bronchi in cav-1 deficient (cav-1−/−) and wild-type mice using organ bath recordings and videomorphometry of methyl-beta-cyclodextrin (MCD) treated and non-treated precision-cut lung slices (PCLS). The presence of caveolae was investigated by electron microscopy. Receptor subtypes driving 5-HT-responses were studied by RT-PCR and videomorphometry after pharmacological inhibition with ketanserin. Cav-1 was present in tracheal epithelium and ASM. Muscarine induced a dose dependent contraction in all airway segments. A significantly higher Emax was observed in the caudal trachea. Although, caveolae abundancy was largely reduced in cav-1−/− mice, muscarine-induced airway contraction was maintained, albeit at diminished potency in the middle trachea, in the caudal trachea and in the bronchus without changes in the maximum efficacy. MCD-treatment of PLCS from cav-1−/− mice reduced cholinergic constriction by about 50%, indicating that cholesterol-rich plasma domains account for a substantial portion of the muscarine-induced bronchoconstriction. Notably, cav-1-deficiency fully abrogated 5-HT-induced contraction of extrapulmonary airways. In contrast, 5-HT-induced bronchoconstriction was fully maintained in cav-1-deficient intrapulmonary bronchi, but desensitization upon repetitive stimulation was enhanced. RT-PCR analysis revealed 5-HT1B, 5-HT2A, 5-HT6, and 5-HT7 receptors as the most prevalent subtypes in the airways. The 5-HT-induced-constriction in PCLS could be antagonized by ketanserin, a 5-HT2A receptor inhibitor. In conclusion, the role of cav-1, caveolae, and cholesterol-rich plasma domains in regulation of airway tone are highly agonist-specific and dependent on airway level. Cav-1 is indispensable for serotonergic contraction of extrapulmonary airways and modulates cholinergic constriction of the trachea and main bronchus. Thus, cav-1/caveolae shall be considered in settings such as bronchial hyperreactivity in common airway diseases and might provide an opportunity for modulation of the constrictor response.

301 Effect of Ascorbic Acid on Aiway Hyperresponsiveness in Bronchial Asthma

IntroductionAscorbic acid has been reported to have a role in the decrease of airway responsiveness in animal models. This data has been based on the regulation of airway tone and modulation of airway reactivity by ascorbic acid. Human studies show that ascorbic acid has a protective effect against the increase in bronchial responsiveness induced by ozone, and nitrogendioxide. We hypothesized that ascorbic acid may attenuate bronchial hyperresponsiveness in bronchial asthma.MethodsWe studied 15 mild asthma patients and 13 healthy non smoker controls. These patients were measured plasma ascorbic acid levels with 2, 4-DNPH (dinitrophenyhydrazine) method and checked methacholine challenge with Chi method before and 1 hour after ascorbic acid intake (3 gm). To assess chronic effect of ascorbic acid on airway responsiveness, these participants were checked again plasm ascorbic acid and methacholine challenge after daily intake of ascorbic acid (1 gm) for 2 weeks.ResultsThere were no significant differences in plasma ascorbic acid levels in asthma patients and controls. Bronchial hyperresponsiveness was decreased after ascorbic acid intake (3 gm) in asthma patients, but not statistacally significant. This decrease was persisted with daily 1 g of ascorbic acid intake for 2 weeks. PC20FEV1 were not correlated to plasma ascorbic acid levels in asthma patients.ConclusionsIn mild bronchial asthma, Airway hyperresponsiveness may be ameliorated by ascorbic acid supplementations. But further studies are necessary to address the question of the effectiveness of ascorbic acid in bronchial asthma.

The role of extracellular-signal regulate kinase (ERK) in the regulation of airway tone in porcine isolated peripheral bronchioles

Extracellular signal regulated kinase (ERK) is known to regulate vascular smooth muscle contraction. However, a role for ERK in airway smooth muscle contraction has yet to be demonstrated conclusively, although contractile agents increase ERK activity in airway smooth muscle. Rather than initiating contraction, ERK could regulate airway tone by interfering with relaxation. Therefore, the aim of this study was to determine whether ERK regulates contraction or relaxation of airway smooth muscle.Segments of porcine peripheral bronchioles were mounted in an isolated tissue bath in Krebs–Henseleit buffer and maintained at 37°C. Cumulative concentration–response curves to histamine, endothelin-1, or the muscarinic agonist carbachol were then carried out in the absence or presence of the MEK inhibitor PD98059. In separate experiments, cumulative concentration response curves to the β-adrenoceptor agonist isoprenaline or the adenylyl cyclase activator forskolin were carried out in the absence or presence of the MEK inhibitors PD98059 or U0126. ERK activity was measured by Western blotting.All three contractile agents increased ERK activity, but the contractile responses were unaffected by PD98059. On the other hand, both PD98059 and U0126 enhanced the relaxations to isoprenaline but not relaxations to the adenylyl cyclase activator forskolin. The enhancement of isoprenaline-induced relaxations with PD98059 was prevented by the K+ channel blocker tetraethylammonium.These data suggest that ERK regulates airway smooth muscle tone by inhibiting β-adrenoceptor-mediated relaxations, rather than an initiation of contraction. The effect on β-adrenoceptor-mediated responses appears to be through a cAMP-independent mechanism, possibly through an interaction with K+ channels.

Reduced rather than enhanced cholinergic airway constriction in mice with ablation of the large conductance Ca2+‐activated K+channel

The unique voltage- and Ca2+-dependent K+ (BK) channel, prominently expressed in airway smooth muscle cells, has been suggested as an important effector in controlling airway contractility. Its deletion in mice depolarized resting membrane potential of tracheal cells, suggesting an increased open-probability of voltage-gated Ca2+ channels. While carbachol concentration-dependently increased the tonic tension of wild-type (WT) trachea, mutant trachea showed a different response with rapid tension development followed by phasic contractions superimposed on a tonic component. Tonic contractions were substantially more dependent on L-type Ca2+ current in mutant than in WT trachea, even though L-type Ca2+ channels were not up-regulated. In the absence of L-type Ca2+ current, half-maximal contraction of trachea was shifted from 0.51 to 1.7 microM. In agreement, cholinergic bronchoconstriction was reduced in mutant lung slices, isolated-perfused lungs and, most impressively, in mutant mice analyzed by body plethysmography. Furthermore, isoprenaline-mediated airway relaxation was enhanced in mutants. In-depth analysis of cAMP and cGMP signaling revealed up-regulation of the cGMP pathway in mutant tracheal muscle. Inhibition of cGMP kinase reestablished normal sensitivity toward carbachol, indicating that up-regulation of cGMP signaling counterbalances for BK channel ablation, pointing to a predominant role of BK channel in regulation of airway tone.

Male sex hormones promote vagally mediated reflex airway responsiveness to cholinergic stimulation

A sex disparity in airway responsiveness to cholinergic stimulation has been observed in laboratory mice in that males are considerably more responsive than females, but the basis for this difference is unclear. In this report, we demonstrate that male sex hormones promote murine airway responsiveness to cholinergic stimulation via vagus nerve-mediated reflex mechanisms. In tissue bath preparations, no sex-based differences were observed in the contractile responses of isolated tracheal and bronchial ring segments to carbachol, indicating that the mechanism(s) responsible for the in vivo sex difference is (are) absent ex vivo. Bilateral cervical vagotomy was found to abolish in vivo airway responsiveness to methacholine in male mice, whereas it did not alter the responses of females, suggesting a regulatory role for male sex hormones in promoting reflex airway constriction. To test this possibility, we next studied mice with altered circulating male sex hormone levels. Castrated male mice displayed airway responsiveness equivalent to that observed in intact females, whereas administration of exogenous testosterone to castrated males restored responsiveness, albeit not to the level observed in intact males. Administration of exogenous testosterone to intact female mice similarly enhanced responsiveness. Importantly, the promotive effects of exogenous testosterone in castrated male and intact female mice were absent when bilateral vagotomy was performed. Together, these data indicate that male sex hormones promote cholinergic airway responsiveness via a vagally mediated reflex mechanism that may be important in the regulation of airway tone in the normal and diseased lung.

Respiratory syncytial virus bronchiolitis and the pathogenesis of childhood asthma.

There is now convincing evidence that children who develop lower respiratory symptoms during infection with respiratory syncytial virus (RSV) in early life are at increased risk of developing asthma-like symptoms during the school years. What determines this association is not well-understood, but increased likelihood of allergic sensitization plays a minor role, if any, as a determinant of post-RSV wheeze. Current evidence suggests that both genetic and environmental factors determine the type of immune response to the acute RSV infection and that this response, in turn, may affect the development of the control mechanisms involved in the regulation of airway tone. Many different cytokines appear to play a role in this acute immune response, including interferon-gamma; interleukins 8, 10 and 12; and cytokines produced by T helper (Th) 1 and Th2 cells. Because asthma is a heterogeneous condition, future studies will need to determine the potentially different role of RSV infection as a risk factor for these different asthma phenotypes. It is likely, however, that strategies for the prevention of RSV infection may play a role in the prevention of the subsequent development of persistent wheezing and asthma-like symptoms in childhood.

Association of beta(2)-adrenoreceptor variants with bronchial hyperresponsiveness.

Because of its involvement in the regulation of airway tone, the beta(2)-adrenoreceptor is considered a candidate for bronchial hyperresponsiveness (BHR) associated with asthma. This notion is supported by several reports that have implicated the chromosomal region 5q31-q33 harboring the gene for the beta(2)-adrenoreceptor in the genetics of asthma and related phenotypes. We performed a population-based association study focusing on BHR as a qualitative trait and omitting other asthma-related phenotypes. From a German population sample of 1,150 individuals we extracted all 152 bronchohyperreactive probands, who were compared with 295 bronchonormoreactive control subjects. All individuals were genotyped for three single nucleotide polymorphisms of the beta(2)-adrenoreceptor gene resulting in variants at the amino acid positions 16, 27, and 164. The genotyping protocol used allowed the determination of haplotypes of these polymorphisms. Whereas no individual polymorphism was associated with BHR, the Gly16/Gln27/Th164 haplotype was significantly underrepresented in the case group indicating a protective effect of this haplotype with regard to BHR. Upon reanalysis by sex a significant association persisted only for female probands.

Inhaled carbon dioxide inhibits lower airway nitric oxide formation in the guinea pig.

The present study addressed the effect of inhaled carbon dioxide on lower airway nitric oxide formation in normoxic anaesthetized guinea pigs. Ventilation with carbon dioxide (1.5, 3, 6, 9 and 12%) induced a concentration-dependent decrease in the basal concentration of nitric oxide in exhaled tracheal air. A maximal reduction in exhaled nitric oxide of approximately 25% was induced by 12% carbon dioxide in inhaled air. Ventilation with positive end-expiratory pressure (7 cmH2O) increased the concentration of exhaled nitric oxide. Inhalation of carbon dioxide had a larger, concentration-dependent, inhibitory effect (maximally 60%) on the lower airway nitric oxide formation induced by ventilation with positive end-expiratory pressure, as compared with the effect on the basal concentration of nitric oxide. The results show that inhaled carbon dioxide suppresses lower airway nitric oxide excretion in the guinea pig. Endogenous carbon dioxide might exert effects through regulation of endogenous nitric oxide formation, for example in the regulation of airway tone or in ventilation-perfusion matching.

Intracellular pH regulates voltage-dependent Ca2+ channels in porcine tracheal smooth muscle cells

Changes in CO2 or in pH modify airway smooth muscle contractility. To investigate the mechanisms involved, we compared K(+)-induced contractions in porcine bronchial rings exposed to different CO2 concentrations and directly measured the effects of changes in intracellular (pHi) or extracellular pH (pHo) on Ca2+ currents (ICa) through voltage-dependent Ca2+ channels (VDC) in porcine tracheal smooth muscle cells. Hypocapnia and hypercapnia caused leftward and rightward shifts, respectively, in the dose-response to K+ (P < 0.05) but did not change the maximum force obtained. Peak ICa (10 mM external Ca2+) elicited by depolarizing pulses from -80 mV was maximal [-265 +/- 12 pA (mean +/- SE), n = 19] at +10 mV. Intracellular acidification decreased the peak ICa at +10 mV from -261 +/- 20 pA to -177 +/- 12 pA (P < 0.05, n = 4), while intracellular alkalinization increased the peak ICa at +10 mV from -302 +/- 27 pA to -368 +/- 26 pA (P < 0.05, n = 4). Changes in pHo had little effect on ICa. There was no shift in the voltage-dependence of induced ICa with any change. We conclude that pHi, but not pHo, directly modulates the entry of Ca2+ into airway smooth muscle cells through VDC. This mechanism may contribute to regulation of airway tone by CO2.

The effect of a nitric oxide synthase inhibitor on the modulation of airway responsiveness in rats.

The possible role of nitric oxide (NO) in the regulation of airway tone remains to be fully explored. In the present study we examined the effect of NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, on airway responsiveness in rats. The effect of L-NAME on endotoxin (lipopolysaccharide; LPS)-induced changes in airway responsiveness was also evaluated. L-NAME (1 mg/kg given intravenously) caused a 33.3 +/- 6.9% increase in blood pressure, but did not influence baseline airway tone or the provocative dose of carbachol causing a 50% increase in pulmonary resistance (RL)(PD50RL). Exposure of F344 rats to LPS induced a transient increase in airway responsiveness at 90 min after exposure, followed by a significant hyporesponsiveness between 9 and 12 h after exposure. L-NAME (1 mg/kg intravenously) did not influence the increase in responsiveness but inhibited the LPS-induced hyporesponsiveness; in LPS-exposed, L-NAME-treated animals, the PD50RL for carbachol was 3.0 +/- 0.1, versus 4.8 +/- 0.3 micrograms/kg in the LPS-exposed, placebo-pretreated group (p < 0.05). The effect of L-NAME was abolished by pretreatment with L-arginine but not with D-arginine. L-NAME did not influence the LPS-induced increase of neutrophils in bronchoalveolar lavage fluid (BALF). These results suggest that in rats, consitutive NO synthesis does not contribute either to basal airway tone or to the basal degree of airway responsiveness, but that inducible NO synthesis mediates endotoxin-induced hyporesponsiveness.