Sensitized Airway Smooth Muscle Research Articles

The loss of DGK protects against allergic airway inflammation and airway hyperresponsiveness

Abstract Asthma is a chronic allergic inflammatory airway disease that is caused by aberrant immune responses to inhaled allergens, which leads to airflow obstruction driven in part by increased sensitivity of airway smooth muscle to contractile agonists, a process known as airway hyperresponsiveness (AHR). The inflammation of allergic asthma is driven by type 2 cytokines released by Th2 CD4+ T cells and group 2 innate lymphoid cells (ILC2) in the lung. Here, we report that targeting DAG kinase zeta (DGKζ), a negative regulator of DAG-mediated cell signaling, protects against allergic asthma. DGKζ knockout (KO) mice exhibited reduced type 2 airway inflammation and were completely resistant to AHR induction in a mouse model of allergic asthma. Surprisingly, the mechanism by which DGKζ protected against airway inflammation and AHR were separable. Targeted deletion of DGKζ in T cells led to decreased type 2 inflammation with no attenuation of AHR. In contrast, conditional deletion of DGKζ in airway smooth muscle cells led to decreased AHR despite no changes in airway inflammation. Importantly, the pharmacological inhibition of DGK provided protection against airway inflammation and AHR in mice, and also reduced bronchoconstriction of human airways. Together, our data suggest that DGKζ is potentially a novel therapeutic target for allergic asthma. Moreover, the targeted deletion of DGKζ reveals that the inflammatory and AHR components of allergic asthma are not as interdependent, as generally believed.

Dynamic Assessment of Ca2+ Sensitivity of Isometric Force in Intact Airway Smooth Muscle Using Phase Loop Plots

A transient elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) triggers a force response in excitation‐contraction coupling. The Ca2+ sensitivity of muscle reflects the relationship between [Ca2+]cyt and force, and in skeletal and cardiac muscle is often defined as the [Ca2+]cyt at which 50% force is generated (ED50). In smooth muscle, Ca2+ sensitivity has been assessed as the ratio of peak force to peak cytosolic [Ca2+]cyt. However, this conventional measurement overlooks the dynamic nature of Ca2+ regulation of force generation. In the present study, in order to provide a dynamic index of Ca2+ sensitivity in airway smooth muscle (ASM), we simultaneously measured [Ca2+]cyt and force and determined their temporal dependence using a phase loop plot. We examined the phase loop plot assessment of Ca2+ sensitivity in ASM under the condition in which the conditional assessment indicated an increase (Calyculin A treatment) in Ca2+ sensitivity. Intact ASM strips were isolated from porcine tracheas obtained from local abattoir. ASM strips were loaded with Fura‐2 AM (Ca2+ fluorescent indicator) and stimulated using 1 mM ACh. Isometric force and [Ca2+]cyt responses were simultaneously measured using a Guth Muscle Research System. ASM strips were exposed to Calyculin A (phosphatase inhibitor; 100 nM for 10 min), which was previously reported to alter static Ca2+ sensitivity. Phase loop plots were calculated from simultaneous [Ca2+]cyt and force responses. The extent of rMLC phosphorylation was determined at different time periods after ACh stimulation using Phos‐tag SDS‐PAGE (SuperSep Phos‐tag; Wako Pure Chemical Industries, Ltd.) and Western blotting. Calyculin A increased ACh‐induced isometric force in ASM strips, but it had no effect on the amplitude of evoked [Ca2+]cyt transients. These results suggested that Calyculin A increased static Ca2+ sensitivity. However, the phase loop plots showed no significant shift in dynamic Ca2+ sensitivity with Calyculin A. These results indicate that the phase loop plot provides a valuable tool to assess dynamic Ca2+ sensitivity of isometric force generation in ASM, which better reflects the complex process of excitation‐contraction coupling in smooth muscle. These results further indicate that dynamic Ca2+ sensitivity of force generation in ASM is independent of rMLC phosphorylation.Support or Funding InformationSupported by NIH grant HL126451 (GCS)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Use of Phase Loop Plots to Assess Ca2+ Sensitivity of Force Generation in Airway Smooth Muscle

In excitation‐contraction coupling, a transient elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) triggers a force response. The Ca2+ sensitivity of muscle reflects the relationship between [Ca2+]cyt and force, and in skeletal and cardiac muscle is often defined as the [Ca2+]cyt at which 50% force is generated. In smooth muscle, Ca2+ sensitivity has been assessed as the ratio of peak force to peak cytosolic [Ca2+]cyt. However, this measurement overlooks the dynamic nature of Ca2+ regulation of force generation. In the present study, in order to provide a dynamic index of Ca2+ sensitivity in airway smooth muscle (ASM), we simultaneously measured [Ca2+]cyt and force and determined their temporal dependence using a phase loop plot. We hypothesized that [Ca2+]cyt/force phase loop plot in ASM would be altered by conditions in which the extent of regulatory myosin light chain (rMLC) phosphorylation was affected.Intact ASM strips were isolated from porcine tracheas obtained from local abattoir. ASM strips, were loaded with Fura‐2 AM (Ca2+ fluorescent indicator) and stimulated using 1 μM ACh. Isometric force and [Ca2+]cyt responses were simultaneously measured using a Guth Muscle Research System. In the first set of experiments we examined the effect of Y27623, a Rho‐kinase inhibitor, and calyculin A, a protein phosphatase inhibitor that affected Ca2+‐induced rMLC phosphorylation. In a second set of experiments, we examined the effect of cytochalasin D (Cyto‐D), an inhibitor of actin polymerization, which reduces force independent of Ca2+‐induced rMLC phosphorylation.As expected, Y27623 decreased ACh‐induced isometric force but had no effect on evoked [Ca2+]cyt transients. Cyto‐D also decreased force with no effects on evoked [Ca2+]cyt. By comparison, calyculin A increased force with no effect on [Ca2+]cyt. Although these results suggest changes in Ca2+ sensitivity, the phase loop plots showed no significant shift indicative of changes in dynamic Ca2+ sensitivity. These results do not support our hypothesis that dynamic Ca2+ sensitivity in ASM is modulated by the extent of rMLC phosphorylation. Under these experimental conditions, it appears that isometric force generation in ASM is not entirely dependent on [Ca2+]cyt.Support or Funding InformationSupported by NIH grant HL126451 (GCS)

On the relationship between response to inhaled methacholine and airway dimensions

Site-specific dosimetry was used to address the hypothesis that, given the airway dimensions of one subject, the airway dimensions of a second subject can be obtained by comparing responses to a modified bronchoconstrictor inhalation-protocol. Fourteen healthy, nonsmoking subjects received a high-resolution computed tomography (HRCT) scan prior to performing a methacholine challenge and measurement of change in forced-expiratory-volume-in-one-second, (∆FEV1). Scale factors of average length (L) and diameter (d) of the first six tracheobronchial airway generations were used to construct typical path lungs. The Multiple Path Particle Dosimetry (MPPD) model calculated surface density of methacholine. Airway circumference change, ∆C/C0, was used to calculate ∆FEV1 in terms of airway smooth muscle sensitivity, K (cm/µg/cm2). Virtual protocols were modeled such that fractional changes in ∆FEV1,“∆Y”, were independent of K. Eight subjects responded to methacholine with ∆FEV1 >3% and Ks from 0.011 to 35.8. For one virtual protocol, the linear relations between ∆Y and length and diameter scale factors had R2s respectively of 0.49 and 0.57. Within the limits of these measurements, the hypothesis is demonstrated; — any continuously increasing function of ∆Y vs. scale factor being sufficient to allow (without HRCT scans) classification of subjects according to airway size.

Airway Smooth Muscle Sensitivity to Methacholine in Precision-Cut Lung Slices (PCLS) from Ovalbumin-induced Asthmatic Mice.

Asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness (AHR) and reversible airway obstruction. Methacholine (MCh) is widely used in broncho-provocation test to evaluate airway resistance. For experimental investigation, ovalbumin-induced sensitization is frequently used in rodents (Ova-asthma). However, albeit the inflammatory histology and AHR in vivo, it remains unclear whether the MCh sensitivity of airway smooth muscle isolated from Ova-asthma is persistently changed. In this study, the contractions of airways in precision-cut lung slices (PCLS) from control, Ova-asthma, and IL-13 overexpressed transgenic mice (IL-13TG) were compared by analyzing the airway lumen space (AW). The airway resistance in vivo was measured using plethysmograph. AHR and increased inflammatory cells in BAL fluid were confirmed in Ova-asthma and IL-13TG mice. In the PCLS from all three groups, MCh concentration-dependent narrowing of airway lumen (ΔAW) was observed. In contrast to the AHR in vivo, the EC50 of MCh for ΔAW from Ova-asthma and IL-13TG were not different from control, indicating unchanged sensitivity to MCh. Although the AW recovery upon MCh-washout showed sluggish tendency in Ova-asthma, the change was also statistically insignificant. Membrane depolarization-induced ΔAW by 60 mM K+ (60K-contraction) was larger in IL-13TG than control, whereas 60K-contraction of Ova-asthma was unaffected. Furthermore, serotonin-induced ΔAW of Ova-asthma was smaller than control and IL-13TG. Taken together, the AHR in Ova-asthma and IL-13TG are not reflected in the contractility of isolated airways from PCLS. The AHR of the model animals seems to require intrinsic agonists or inflammatory microenvironment that is washable during tissue preparation.

Relaxant effect of superimposed length oscillation on sensitized airway smooth muscle.

Asthma is associated with reductions in the airway lumen and breathing difficulties that are attributed to airway smooth muscles (ASM) hyperconstriction. Pharmaceutical bronchodilators such as salbutamol and isoproterenol are normally used to alleviate this constriction. Deep inspirations and tidal oscillations (TO) have also been reported to relax ASM in healthy airways with less response in asthmatics. Little information is available on the effect of other forms of oscillation on asthmatic airways. This study investigates the effect of length oscillations (LO), with amplitude 1 and 1.5% in the frequency range 5-20 Hz superimposed on breathing equivalent LO, on contracted ASM dissected from sensitized mice. These mice are believed to show some symptoms such as airway hyperreactivity similar to those associated with asthma in humans. In the frequency range used in this work, this study shows an increase in ASM relaxation of an average of 10% for 1.5% amplitude when compared with TO, ISO, or the combination of both. No similar finding is observed with 1% amplitude. This suggests that superimposed length oscillation acting over the interaction of myosin and actin during contraction may lead to temporal rearrangement and disturbance of the cross-bridge process in asthmatic airways.

Ca2+ handling and sensitivity in airway smooth muscle: Emerging concepts for mechanistic understanding and therapeutic targeting

Free calcium ions within the cytosol serve as a key secondary messenger system for a diverse range of cellular processes. Dysregulation of cytosolic Ca2+ handling in airway smooth muscle (ASM) has been implicated in asthma, and it has been hypothesised that this leads, at least in part, to associated changes in both the architecture and function of the lung. Significant research is therefore directed towards furthering our understanding of the mechanisms which control ASM cytosolic calcium, in addition to those regulating the sensitivity of its downstream effector targets to calcium. Key aspects of the recent developments in this field were discussed at the 8th Young Investigators' Symposium on Smooth Muscle (2013, Groningen, The Netherlands), and are outlined in this review.

Phosphodiesterase 4 Inhibitor Roflumilast Improves the Bronchodilative Effect of Sevoflurane in Sensitized Airways

Although phosphodiesterase 4 inhibitors and the volatile anesthetic sevoflurane are known to have independent bronchodilator properties, the combined administration of these two agents may have the potential to exert an additive or synergistic bronchodilator effect. The authors tested this hypothesis and investigated the common site of this combined relaxation effect in a model of airway hyperresponsiveness with ovalbumin-sensitized guinea pigs. Ovalbumin-sensitized animals (n = 138) were randomized into six groups: sensitized, sevoflurane, rolipram1.0, roflumilast1.0, sevoflurane/rolipram1.0, and sevoflurane/roflumilast1.0. Total lung resistance in vivo, airway smooth muscle tension in vitro, and intracellular cyclic adenosine monophosphate levels were measured to evaluate the relaxation effect. Among the six sensitized groups, total lung resistance was higher in the order of sensitized > sevoflurane > rolipram 1.0 > roflumilast1.0 > sevoflurane/rolipram1.0 > sevoflurane/roflumilast1.0, with an increase in acetylcholine concentration. Compared with the other five groups, the muscle tensions in the sevoflurane/roflumilast1.0 group were significantly lower at carbacholine doses of 10, 10, and 10 M; the cyclic adenosine monophosphate concentrations (means ± SD) in the sevoflurane/rolipram1.0 (1.61 ± 0.34) and sevoflurane/roflumilast1.0 (1.50 ± 0.20) groups were higher than that in the sensitized (0.52 ± 0.15) and sevoflurane (1.12 ± 0.32) groups. The combined use of phosphodiesterase 4 inhibitors with the volatile anesthetic sevoflurane had an additive bronchodilator effect in ovalbumin-sensitized guinea pigs. The concurrent increase in cyclic adenosine monophosphate levels in sensitized airway smooth muscle might be a mechanism of this combined relaxation effect.

Hyperresponsiveness to adenosine in sensitized Wistar rats over-expressing A1 receptor

Airway hyperreactivity is characterized by increased responsiveness to bronchoconstrictor stimuli and it is hallmark of asthma. Adenosine is an ubiquitous signaling nucleoside resulting from ATP catabolism, whose extracellular levels increase following cellular damage or stress. Adenosine plays a role in asthma; asthmatics, but not normal subjects, present bronchoconstriction following inhalation of adenosine or of its precursor, adenosine-5′-monophosphate, most likely via adenosine A2B receptor on mast cells. However, the mechanism underling the increased airway smooth muscle sensitivity to adenosine in asthmatics remains to be elucidated. Early experimental studies suggested the involvement of A1 receptor; this hypothesis has been confirmed by more recent studies on guinea pigs and is corroborated by the finding of an increased adenosine A1 expression on asthmatic bronchial tissues. Brown Norway rats, the strain usually used to assess asthma models, develop hyperresponsiveness to adenosine 3h following allergen challenge, but not 24h thereafter, without involvement of A1 receptor. Here, we investigated the role of adenosine A1 receptor in sensitized Wistar rats showing airway hyperresponsiveness 24h following allergen challenge. We found that on bronchi of sensitized Wistar rats challenged with allergen there is an increased adenosine A1 receptor expression on smooth muscle that is responsible for hyperresponsiveness to adenosine and ovalbumin.

Role of Sphingosine-1-Phosphate in β-adrenoceptor Desensitization via Ca2+ Sensitization in Airway Smooth Muscle

The correlation between inflammatory cells and airway smooth muscle plays fundamental roles in the pathophysiology of asthma. This study was designed to determine whether pre-exposure of airway smooth muscle to sphingosine-1-phosphate (S1P), which is released from mast cells by allergic reactions, causes a deterioration of β-adrenoceptor function. Isometric tension and the ratio of fluorescence intensities at 340 and 380 nm (F(340)/F(380)), an indicator of intracellular Ca2+ levels, were simultaneously measured using fura-2 loaded guinea-pig tracheal tissues. Intracellular cAMP levels were also measured. Pre-exposure to S1P caused a reduction in the inhibitory effects of 0.3μM isoprenaline, a β-adrenoceptor agonist, and 10μM forskolin, a direct activator of adenylyl cyclase, against 1μM methacholine-induced contraction in concentration- and time- dependent manners. In contrast, the values of F(340)/F(380) were not augmented under this experimental condition. After incubation with S1P in the presence of 0.001-1μM Y-27632, a Rho-kinase inhibitor, the reduced responsiveness to forskolin induced by S1P was reversed in a concentration-dependent manner. Moreover, pre-treatment with pertussis toxin (PTX), an inhibitor of G(i), suppressed the loss of forskolin-induced relaxation induced by S1P. Pre-exposure to S1P markedly inhibited the augmentation of cAMP accumulation induced by forskolin. However, addition of Y-27632 and pre-exposure to PTX returned forsokin-induced cAMP accumulation to the control level. Pre-exposure to S1P causes heterologus desensitization of β-adrenoceptors by increasing the sensitivity of airway smooth muscle to intracellular Ca2+. Ca2+ sensitization regulated by G(i) and Rho-kinase is involved in this phenomenon.

Involvement of IL-13 in Tobacco Smoke–Induced Changes in the Structure and Function of Rat Intrapulmonary Airways

Chronic obstructive pulmonary disease (COPD) involves disease of small airways with an increase in airway smooth muscle sensitivity to spasmogens and with structural changes described as airway remodeling. We investigated the effect of tobacco smoke (TS) exposure on the structure and function of small airways in rats and the role of IL-13 in this response. Precision-cut lung slices (230-280 microm) were prepared from male Sprague-Dawley rats after acute (3 d) or chronic (8 or 16 wk) daily exposure to TS or air. Carbachol (CCh) and 5-hydroxytryptamine (5HT) concentration responses were performed on airways (50-400 microm diameter). The effect of IL-13 in vitro on small airway sensitivity to CCh and 5HT was also determined. Acute exposure to TS did not affect the sensitivity of the intrapulmonary airways to either spasmogen. After 8 weeks of TS exposure, airway hyperresponsiveness (AHR) to CCh was evident (log EC(50) CCh: air = 0.22 microM; TS = -0.12 microM; P = 0.019); AHR to 5HT was also observed after the 16-week exposure to TS (air = -0.85 microM; TS = -1.06 microM; P = 0.038). Chronic TS exposure increased airway wall SMA content, which correlated with increased expression of IL-13 and transforming growth factor (TGF)-beta(1) in the lung tissues. In vitro incubation with IL-13, but not TGF-beta(1), induced changes in small airway sensitivity to CCh and 5HT. Chronic TS exposure induces increased responsiveness in intrapulmonary airways of rats that may be mediated in part by an increase in IL-13.

Sensitized airway smooth muscle plasticity and hyperreactivity: a reviewThis article is one of a selection of papers published in the Special Issue on Recent Advances in Asthma Research.

To help elucidate the mechanisms underlying asthmatic bronchospasm, the goal of our research has been to determine whether airway smooth muscle (ASM) hyperreactivity was the responsible factor. We reported that in a canine model of asthma, the shortening capacity (DeltaLmax) and velocity (Vo) of in vitro sensitized muscle were significantly increased. This increase was of sufficient magnitude to account for 75% narrowing of the in vivo airway, but maximal isometric force was unchanged. This last feature has been reported by others. Under lightly loaded conditions, ASM completes 75% of its isotonic shortening within the first 2 s. Furthermore, 90% of the increased shortening of ragweed pollen-sensitized ASM (SASM), compared with control (CASM), is complete within the first 2 s. The study of shortening beyond this period will apparently not yield much useful information, and studies of isotonic shortening should be focused on this interval. Although both CASM and SASM showed plasticity and adaptation with respect to isometric force, neither muscle type showed a difference in the force developed in these phases. During isotonic shortening, no evidence of plasticity was seen, but the equilibrated SASM showed increased DeltaLmax and Vo of shortening. Molecular mechanisms of changes in Vo could result from changes in the kinetics of the myosin heavy chain ATPase. Motility assay, however, showed no changes between CASM and SASM in the ability of the purified myosin molecule (SF1) to translocate a marker actin filament. On the other hand, we found that the state of activation of the ATPase by phosphorylation of smooth muscle myosin light chain (molecular mass 20,000 Da) was greater in the SASM. This would account for the increased Vo. Investigating the signalling pathway, we found that whereas [Ca2+]i increased in both isometric and isotonic contraction, there was no significant difference between CASM and SASM. The content and activity of calmodulin were also not different between the 2 muscles. Nevertheless, we did find that content and total activity of smooth muscle myosin light chain kinase (smMLCK) and the abundance of its message were greater; this would explain the increased MLC20 phosphorylation. The binding affinity between Ca2+ and calmodulin and between 4 Ca2+ calmodulin and smMLCK remains to be studied. We conclude that SASM shows increased isotonic shortening capacity and velocity. It also shows increased content and total activity of smMLCK, which is consistent with the increased shortening. Plasticity produced by oscillation is not seen in the shortening muscle, although it is seen with respect to force development. It did not modulate the behaviour of the sensitized muscle.

Inhibition of Rho-kinase normalizes nonspecific hyperresponsiveness in passively sensitized airway smooth muscle preparations

Currently, little is known about mechanisms underlying passive sensitization-induced nonspecific airway hyperresponsiveness. We sought to determine whether the nonspecific airway hyperresponsiveness observed after passive sensitization involves an increased role of Rho-kinase in airway smooth muscle contraction. In addition, the contribution of Rho-kinase to specific allergen-induced airway smooth muscle contraction was studied. Guinea pig tracheal smooth muscle preparations were incubated for 16 h, in the presence of serum obtained from nonsensitized guinea pigs or atopic serum obtained from actively ovalbumin-sensitized guinea pigs. After incubation, the contribution of Rho-kinase to histamine-, methacholine- or ovalbumin-induced isometric contractions was determined, using the specific Rho-kinase inhibitor Y-27632. Maximal contractions induced by histamine and methacholine were significantly increased in passively sensitized preparations, without a change in potency (− logEC 50). In control preparations, Y-27632 reduced the potency of both agonists, without affecting maximal contraction. Remarkably, the increased agonist responsiveness induced by passive sensitization was fully normalized by Y-27632. Treatment with Y-27632 also reduced ovalbumin-induced contraction in these preparations. This study shows that the nonspecific airway smooth muscle hyperresponsiveness as well as the specific allergen responsiveness induced by passive sensitization are dependent on Rho-kinase. The complete inhibition by Y-27632 of the passive sensitization-induced increased responsiveness toward histamine and methacholine indicates a pivotal role of Rho-kinase in this process.

Isotonic relaxation of control and sensitized airway smooth muscle

Smooth muscle relaxation has most often been studied in isometric mode. However, this only tells us about the stiffness properties of the bronchial wall and thus only about wall capacitative properties. It tells us little about airflow. To study the latter, which of course is the meaningful parameter in regulation of ventilation and in asthma, we studied isotonic shortening of bronchial smooth muscle (BSM) strips. Failure of BSM to relax could be another important factor in maintaining high airway resistance. To analyze relaxation curves, we developed an index of isotonic relaxation, t1/2(P, lCE), which is the half-time for relaxation that is independent of muscle load (P) and of initial contractile element length (lCE). This index was measured in curves of relaxation initiated at 2 s (normally cycling crossbridges) and at 10 s (latch-bridges). At 10 s no difference was seen for adjusted t1/2(P, lCE) between curves obtained from control and sensitized BSM, (8.38 +/- 0.92 s vs. 7.78 +/- 0.93 s, respectively). At 2 s the half-time was almost doubled in the sensitized BSM (6.98 +/- 0.01 s (control) vs. 12.74 +/- 2.5 s (sensitized)). Thus, changes in isotonic relaxation are only seen during early contraction. Using zero load clamps, we monitored the time course of velocity during relaxation and noted that it varied according to 3 phases. The first phase (phase i) immediately followed cessation of electrical field stimulation (EFS) at 10 s and showed almost the same velocity as during the latter 1/3 of shortening; the second phase (phase ii) was linear in shape and is associated with zero load velocity, we speculate it could stem from elastic recoil of the cells' internal resistor; and the third phase (phase iii) was convex downwards. The zero load velocities in phase iii showed a surprising spontaneous increase suggesting reactivation of the muscle. Measurements of intracellular calcium (Fura-2 study) and of phosphorylation of the 20 kDa myosin light chain showed simultaneous increments, indicating phase iii represented an active process. Studies are under way to determine what changes occur in these 3 phases in a sensitized muscle. And of course, in the context of this conference, just what role the plastic properties of the muscle play in relaxation requires serious consideration.

Anesthetics Inhibit Membrane Receptor Coupling to the Gq/11Heterotrimeric G Protein in Airway Smooth Muscle

Some anesthetics relax airway smooth muscle in part by inhibiting acetylcholine-induced increases in Ca2+ sensitivity, an effect associated with inhibition of guanosine nucleotide exchange at the alpha subunit of the Gq/11 (Galphaq/11) heterotrimeric G protein. This study tested the hypothesis that these anesthetic effects are not unique to the muscarinic receptor but are a general property of the heptahelical receptors that increase Ca2+ sensitivity in airway smooth muscle. Anesthetic effects on agonist-induced increases in Ca2+ sensitivity were measured in porcine airway smooth muscle strips permeabilized with S. aureus alpha-toxin. Anesthetic effects on basal (without agonist stimulation) and agonist-promoted Galphaq/11 guanosine nucleotide exchange were determined in crude membranes prepared from porcine airway smooth muscle. The nonhydrolyzable, radioactive form of guanosine 5'-triphosphate was used as the reporter for nucleotide exchange at Galphaq/11. Acetylcholine, endothelin-1, and histamine caused a concentration-dependent increase in Ca2+ sensitivity. Halothane (0.67 +/- 0.07 mM) and hexanol (10 mM) significantly inhibited the increase in Ca2+ sensitivity induced by each agonist. Each agonist also caused a time- and concentration-dependent increase in Galphaq/11 nucleotide exchange. Neither anesthetic had an effect on basal Galphaq/11 nucleotide exchange, whereas halothane and hexanol significantly inhibited the increase in Galphaq/11 nucleotide exchange promoted by each agonist. These data suggest that inhibition of agonist-promoted guanosine nucleotide exchange at Galphaq/11 by some anesthetics may be a general property of heptahelical receptors involved cellular processes mediated by Galphaq/11, including muscarinic, endothelin-1, and histamine receptor activation of Ca2+ sensitivity.

Asian pear pectin administration during presensitization inhibits allergic response to ovalbumin in BALB/c mice.

A type of respiratory disorder resembling some aspects of human allergic asthma can be induced in mice using ovalbumin. The factors that influence the etiology of asthma are poorly understood even though cytokines are known to play a pivotal role. The purpose of this study was to test the hypothesis whether an administration of Asian pear pectin during presensitization could suppress allergic response to ovalbumin in BALB/c mice. High-dose (100 microg) of pectin-sol was used and values were compared to those from the control. Ovalbumin and aluminum hydroxide were utilized for sensitization while ovalbumin aerosol was used for provocation 2 weeks later. The bronchoalveolar lavage (BAL) and assessment of tracheal smooth muscle responsiveness to electrical field stimulation or acetylcholine were performed 1 day after ovalbumin provocation. Two main cytokines of interferon (IFN)-gamma and interleukin (IL)-5, and serum immunoglobulin E (IgE) were assayed. Laboratory of the Chosun University Medical School Male BALB/c mice Antigen dose of 5 microg for sensitization generated TH1 type cytokines in the lungs with a high level of IFN-gamma and a low level of IL-5. In contrast, TH2 type cytokines were produced in splenocytes including a high level of IL-5 and a low level of IFN-gamma. Asian pear pectin-sol administration during presensitization significantly inhibited (p < 0.05) sensitivity of airway smooth muscle to electrical field stimulation and acetylcholine. Further, IFN-gamma production significantly decreased (p < 0.05) in BAL fluids while it significantly increased (p < 0.05) in splenic cells. On the other hand, IL-5 production significantly increased (p < 0.05) in BAL fluids while it was a significant decrease (p < 0.05) in splenic cells. For the histopathologic changes in the lung, pear pectin-sol recovered ovalbumin (OVA)-induced abnormal signs to an almost normal state. As a correlate, IgE production significantly decreased (p < 0.05) in pectin-sol-treated animals compared to the control. It is possible from these data that BALB/c mice have different susceptibilities to different doses of OVA regulated by pulmonary TH1 and TH2 type cytokines, independent of splenic TH1 and TH2 type cytokines production. These results also indicate that administration of Asian pear pectin-sol in presensitized mice suppresses allergic asthmatic reaction.

Dual effects of hexanol and halothane on the regulation of calcium sensitivity in airway smooth muscle.

Contraction of airway smooth muscle is regulated by receptor-coupled mechanisms that control the force developed for a given cytosolic calcium concentration (i.e., calcium sensitivity). Halothane antagonizes acetylcholine-induced increases in calcium sensitivity by inhibiting GTP-binding (G)-protein pathways. The authors tested the hypothesis that hexanol, like halothane, inhibits agonist-induced increases in calcium sensitivity in airway smooth muscle by inhibiting G-protein pathways. Calcium sensitivity was assessed using alpha-toxin-permeabilized canine tracheal smooth muscle. In selected experiments, regulatory myosin light chain phosphorylation was also determined by Western blotting in the presence and absence of 10 mm hexanol and/or 100 microm acetylcholine. Hexanol (10 mm) and halothane (0.76 mm) attenuated acetylcholine-induced calcium sensitization by decreasing regulatory myosin light chain phosphorylation during receptor stimulation. Hexanol also inhibited increases in calcium sensitivity due to direct stimulation of heterotrimeric G-proteins with tetrafluoroaluminate but not with 3 microm GTPgammaS, consistent with prior results obtained with halothane. In contrast, in the absence of receptor stimulation, both compounds produced a small increase in calcium sensitivity by a G-protein-mediated increase in regulatory myosin light chain phosphorylation that was not affected by pertussis toxin treatment. The authors noted dual effects of hexanol and halothane. In the presence of muscarinic receptor stimulation, hexanol, like halothane, decreases calcium sensitivity by interfering with heterotrimeric G-protein function. However, in the absence of muscarinic receptor stimulation, hexanol and halothane slightly increase calcium sensitivity by a G-protein-mediated process not sensitive to pertussis toxin. Hexanol may represent a useful experimental tool to study the effect of anesthetics on heterotrimeric G-protein function.

A novel mechanism by which hydrogen peroxide decreases calcium sensitivity in airway smooth muscle.

The purpose of this study was to test the hypothesis that H(2)O(2) decreases the amount of force produced by a given intracellular Ca(2+) concentration (i.e., the Ca(2+) sensitivity) in airway smooth muscle (ASM) in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. A new preparation was developed and validated in which canine ASM strips were first exposed to H(2)O(2) and then permeabilized with 10% Triton X-100 to assess the persistent effects of H(2)O(2) on Ca(2+) sensitivity. Experiments in which H(2)O(2) was administered before permeabilization revealed a novel mechanism that contributed to reduced Ca(2+) sensitivity independently of changes in rMLC phosphorylation, in addition to an rMLC phosphorylation-dependent mechanism. The mechanism depended on factors not available in the permeabilized ASM strip or in the buffer to which the strip was exposed, since there was no effect when H(2)O(2) was added to permeabilized strips. H(2)O(2) treatment of a maximally thiophosphorylated purified myosin subfragment (heavy meromyosin) significantly reduced actomyosin ATPase activity, suggesting one mechanism by which the phosphorylation-independent reduction in Ca(2+) sensitivity may occur.

Differential effects of soluble and particulate guanylyl cyclase on Ca2+sensitivity in airway smooth muscle

Maximal relaxation of airway smooth muscle (ASM) in response to atrial natriuretic peptide (ANP), which stimulates particulate guanylyl cyclase (pGC), is less than that produced by nitric oxide (NO) and other compounds that stimulate soluble guanylyl cyclase (sGC). We hypothesized that stimulation of pGC relaxes ASM only by decreasing intracellular Ca(2+) concentration ([Ca(2+)](i)), whereas stimulation of sGC decreases both [Ca(2+)](i) and the force developed for a given [Ca(2+)](i) (i.e., the Ca(2+) sensitivity) during muscarinic stimulation. We measured the relationship between force and [Ca(2+)](i) (using fura 2) under control conditions (using diltiazem to change [Ca(2+)](i)) and during exposure to ANP, diethylamine-NO (DEA-NO), sodium nitroprusside (SNP), and the Sp diastereoisomer of beta-phenyl-1,N(2)-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothionate (Sp-8-Br-PET-cGMPS), a cell-permeant analog of cGMP. Addition of DEA-NO, SNP, or Sp-8-Br-PET-cGMPS decreased both [Ca(2+)](i) and force, causing a significant rightward shift of the force-[Ca(2+)](i) relationship. In contrast, with ANP exposure, the force-[Ca(2+)](i) relationship was identical to control, such that ANP produced relaxation solely by decreasing [Ca(2+)](i). Thus, during muscarinic stimulation, stimulation of pGC relaxes ASM exclusively by decreasing [Ca(2+)](i), whereas stimulation of sGC decreases both [Ca(2+)](i) and Ca(2+) sensitivity.

Autocrine signaling by IL-10 mediates altered responsiveness of atopic sensitized airway smooth muscle.

To elucidate the role and mechanism of action of interleukin (IL)-10 in regulating airway smooth muscle (ASM) responsiveness in the atopic asthmatic state, isolated rabbit tracheal ASM segments were passively sensitized with serum from atopic asthmatic patients or nonatopic nonasthmatic (control) subjects in both the absence and presence of an anti-IL-10 receptor blocking antibody (Ab). Relative to control ASM, atopic asthmatic serum-sensitized tissues exhibited enhanced isometric constrictor responses to administered acetylcholine and attenuated the relaxation responses to isoproterenol. These proasthmatic effects were prevented in atopic asthmatic serum-sensitized ASM that was pretreated with anti-IL-10 receptor Ab. In complementary experiments, exposure of cultured human ASM cells to atopic asthmatic serum induced upregulated expression of IL-10 mRNA. Moreover, extended studies demonstrated that 1) exogenous IL-10 administration to naive ASM elicited augmented contractility to acetylcholine and impaired relaxation to isoproterenol, 2) these effects of IL-10 were prevented by pretreating the tissues with an IL-5 receptor Ab, and 3) IL-10 administration induced upregulated mRNA expression and release of IL-5 protein from cultured ASM cells. Collectively, these findings provide new evidence demonstrating that the altered responsiveness of atopic asthmatic serum-sensitized ASM is largely attributed to activation of an intrinsic T helper type 2-type autocrine mechanism involving IL-10-mediated release and the action of IL-5 in the sensitized ASM itself.