Tracheal Strips Research Articles

Sensitivity of the airway smooth muscle in terms of force, shortening and stiffness

Eight pig tracheal strips were stimulated to contract with log increments of methacholine from 10-8 to 10-5 M. For each strip, the concentration-response was repeated four times in a randomized order to measure isometric force, isotonic shortening against a load corresponding to either 5 or 10 % of a reference force, and average force, stiffness, elastance and resistance over one cycle while the strip length was oscillating sinusoidally by 5 % at 0.2 Hz. For each readout, the logEC50 was calculated and compared. Isotonic shortening with a 5 % load had the lowest logEC50 (-7.13), yielding a greater sensitivity than any other contractile readout (p<0.05). It was followed by isotonic shortening with a 10 % load (-6.66), elastance (-6.46), stiffness (-6.46), resistance (-6.38), isometric force (-6.32), and average force (-6.30). Some of these differences were significant. For example, the EC50 with the average force was 44 % greater than with the elastance (p=0.001). The methacholine sensitivity is thus affected by the contractile readout being measured.

Discovery and Identification of Novel 5-Hydroxy-4H-benzo[1,4]oxazin-3-one Derivatives as Potent β2-Adrenoceptor Agonists through Structure-Based Design, Synthesis, and Biological Evaluation.

Although β2-agonists are crucial for treatment of chronic respiratory diseases, optimizing β2-agonistic activity and selectivity remains essential for achieving favorable therapeutic outcomes. A structure-based molecular design workflow was employed to discover a novel class of β2 agonists featuring a 5-hydroxy-4H-benzo[1,4]oxazin-3-one scaffold, which potently stimulated β2 adrenoceptors (β2-ARs). Screening for the β2-agonistic activity and selectivity led to the identification of compound A19 (EC50 = 3.7 pM), which functioned as a partial β2-agonist in HEK-293 cells containing endogenous β2-ARs. Compound A19 exhibited significant relaxant effects, rapid onset time (Ot50 = 2.14 min), and long duration of action (>12 h) on isolated guinea pig tracheal strips, as well as advantageous pharmacokinetic characteristics in vivo, rendering A19 suitable for inhalation administration. Moreover, A19 suppressed the upregulation of inflammatory cytokines and leukocytes and improved lung function in a rat model of COPD, thereby indicating that A19 is a potential β2 agonist candidate for further study.

A Novel Role of Uricosuric Agent Benzbromarone in BK Channel Activation and Reduction of Airway Smooth Muscle Contraction.

The uricosuric drug benzbromarone, widely used for treatment of gout, hyperpolarizes the membrane potential of airway smooth muscle cells, but how it works remains unknown. Here we show a novel role of benzbromarone in activation of large conductance calcium-activated K+ channels. Benzbromarone results in dose-dependent activation of macroscopic big potassium (BK) currents about 1.7- to 14.5-fold with an EC50 of 111 μM and shifts the voltage-dependent channel activation to a more hyperpolarizing direction about 10 to 54 mV in whole-cell patch clamp recordings. In single-channel recordings, benzbromarone decreases single BKα channel closed dwell time and increases the channel open probability. Coexpressing β1 subunit also enhances BK activation by benzbromarone with an EC50 of 67 μM and a leftward shift of conductance-voltage (G-V) curve about 44 to 138 mV. Site-directed mutagenesis reveals that a motif of three amino acids 329RKK331 in the cytoplasmic linker between S6 and C-terminal regulator of potassium conductance (RCK) gating ring mediates the pharmacological activation of BK channels by benzbromarone. Further ex vivo assay shows that benzbromarone causes reduction of tracheal strip contraction. Taken together, our findings demonstrate that uricosuric benzbromarone activates BK channels through molecular mechanism of action involving the channel RKK motif of S6-RCK linker. Pharmacological activation of BK channel by benzbromarone causes reduction of tracheal strip contraction, holding a repurposing potential for asthma and pulmonary arterial hypertension or BK channelopathies. SIGNIFICANCE STATEMENT: We describe a novel role of uricosuric agent benzbromarone in big potassium (BK) channel activation and relaxation of airway smooth muscle contraction. In this study, we find that benzbromarone is an activator of the large-conductance Ca2+- and voltage-activated K+ channel (BK channel), which serves numerous cellular functions, including control of smooth muscle contraction. Pharmacological activation of BK channel by the FDA-approved drug benzbromarone may hold repurposing potential for treatment of asthma and pulmonary arterial hypertension or BK channelopathies.

Evaluation of the Antiasthmatic Activity of Carissa opaca in Animal Models.

Carissa opaca Stapf ex Haines (C. opaca) fruit is used traditionally in the treatment of respiratory illnesses including asthma. However, there is no scientific evidence supporting its antiasthmatic activity. The current study was conducted to evaluate its antiasthmatic effects using in vivo and in vitro approaches. The methanolic crude extract of C. opaca fruit (Co.Cr.) was used and in vivo antiasthmatic activity was carried out using ovalbumin- (OVA-) sensitized and OVA-challenged BALB/c mice. In in vitro bronchorelaxant activity of crude extract, aqueous and n-hexane fractions of C. opaca were carried out on isolated rat tracheal strips. Co.Cr. (200 and 400 mg/kg) attenuated ovalbumin-induced changes in lung histochemistry with % decrease in peribronchial inflammation of 14.1 ± 0.21 and 65.8 ± 0.22 and % decrease in total inflammatory cell count of 35.7 ± 2.80 and 53.3 ± 2.30 in bronchoalveolar lavage fluid. Co.Cr., aqueous, and n-hexane fraction of C. opaca attenuated the precontractions induced by high K+ (80 mM) and carbachol (1 μM), respectively. In conclusion, the results showed that C. opaca possesses antiasthmatic activity via relaxant effect on bronchial smooth muscle which is mediated through calcium channel blockade and antimuscarinic activity. This study provides scientific evidence of the traditional use of C. opaca in the management of allergic asthma.

STAMINA A NOVEL PROSPECT IN ASTHMA MANAGEMENT

Background: Current therapy for asthma includes bronchodilators and anti-inflammatory drugs. Inhibition ofRhoA/ROCK pathway to control hyperresponsiveness presents a novel area in asthma therapy.Study Design: Experimental randomized control trial. “To examine the effect of statins on the airway hyperresponsiveness.” Statins are lipid-lowering agents exhibiting pleiotropic effects in decreasing oxidative stress and inflammation. Several published studies have reported using statins in treating asthma patients, but their results could bemore consistent. This study aims to determine whether statins are beneficial for asthma administration and explorethe covariables that may affect their clinical effectiveness.Materials and Maths: Five groups of six similar guinea pigs animals under similar laboratory conditions wereselected. The animal model of asthma was reproduced via intraperitoneal ova albumin in all except negative controlgroup 1. These groups were Group I negative control, II positive- control, III atorvastatin treated, IV Simvastatintreated, and V lovastatin treated group. After 28 days, animals were sacrificed. Bronchoalveolar fluid (B.A.L.)analysis was taken, and Smooth muscle contractions in tracheal strips to increasing concentrations of acetylcholinewere recorded by isometric transducers using a computerized data recording system. The effects of statins were assessedby comparing concentration-response curves of experimental groups with those of controls.A systematic literature search was performed in PubMed, Embase, and Cochrane Center Register of ControlledTrials from inception to September 2012. Randomized controlled trials (R.C.T.), retrospective studies, and controlledclinical trials that reported the use of statins in the treatment of asthma patients were eligible. Quality evaluation wasconducted for R.C.T. using Jadad criteria.Results: Comparison of B.A.L. of positive control with that of negative confirmed the induction of allergic inflammatory model. The concentration-response curve was significantly shifted upward in positive control compared to negativecontrol (P&lt;0.05).In the case of statin-treated groups, there was a significant downward shift in concentration-responsecurves compared to positive control, showing inhibition of airways hyperresponsiveness by statins. Significant differenceswere observed between them and positive control (P&lt;0.05A total of 18 articles were included. Our study found noconclusive evidence to demonstrate that statins could enhance lung function in asthmatics, although they may reduceairway inflammation. The results were inconsistent across studies concerning symptoms, quality of life, maintenancemedication, and asthma hospitalization/emergency department (E.D.) visits. The present study demonstrates thatstatins inhibit hyperresponsiveness in the allergic airway inflammatory model of the guinea pig. Statins may have atherapeutic potential to ameliorate airway hyperresponsiveness in allergic bronchial asthma. Human trials are neededto include these drugs in asthma treatment guidelinesKeywords: Asthma Guinea pig Model Statin B.A.L

8-Hydroxyquinolin-2(1H)-one analogues as potential β2-agonists: Design, synthesis and activity study.

β2-Agonists that bind to plasmalemmal β2-adrenoceptors causing cAMP accumulation are widely used as bronchodilators in chronic respiratory diseases. Here, we designed and synthesized a group of 8-hydroxyquinolin-2(1H)-one analogues and studied their β2-agonistic activities with a cellular cAMP assay. Compounds B05 and C08 were identified as potent (EC50<20 pM) and selective β2-agonists among the compounds tested. They behaved as partial β2-agonists in non-overexpressed HEK293cells, and possessed rapid smooth muscle relaxant actions and long duration of action in isolated guinea pig tracheal strip preparations. In summary, B05 and C08 are β2-agonists with potential applicability in chronic respiratory diseases.

Protective Effect of Nedocromil Against Insulin Induced Airway Hyper-Reactivity

Background: Use of inhalable insulin is limited because it causes airway hyper-reactivity. So present study was designed to ameliorate inhalational insulin induced airway hyper-responsiveness.Objectives: The objective of the study was to evaluate the acute effects of insulin on airway reactivity and protective effects of nedocromil against insulin induced airway hyper-reactivity on isolated tracheal tissues of guinea pigs in vitro.Material and Methods: This experimental study was carried out in Pharmacology department of Army Medical College Rawalpindi from January 2012 to July 2012. We observed acute effect of insulin (10-7- 10-3 M) and insulin pretreated with nedocromil (10-5 M) on isolated tracheal strip of guinea pig (n=6) in vitro by constructing cumulative concentration response curves. The tracheal smooth muscle contractions were recorded with Transducer on Four Channel Oscillograph.Results: Insulin significantly increased the tracheal smooth muscle contraction. The mean ± SEM of maximum amplitudes of contraction with insulin and insulin pretreated with nedocromil were 35 ± 1.13 mm and 27.8 ± 1.27 mm respectively. So nedocromil significantly antagonized insulin elicited contractile effect. Conclusion: Nedocromil significantly inhibited the insulin mediated airway hyper-reactivity in guinea pigs. So we suggest that pretreatment of inhaled insulin with nedocromil may have clinical implication in amelioration of its potential respiratory adverse effects.

Effect of Roflumilast in airways disorders via dual inhibition of phosphodiesterase and Ca2+-channel

The bronchodilator effects of Roflumilast “a selective phosphodiesterase type-4 (PDE4)” inhibitor studied in this experimental protocol. The spiral strips of isolated guinea-pig tracheal chains mounted in organ bath and maintained in Krebs solution ventilated with carbogen at 32 °C and in Ca++ restricted krebs solution. PDE inhibitory activity was evaluated by recording dose response curves using inhibitory effect of isoprenaline on CCh induced contractions. For confirmation of PDE inhibition the intracellular cAMP levels were also estimated. Roflumilast resulted a sharp inhibition in contractile responses of carbachol (CCh, 1 µM) and K+ (80 mM) and the results were almost similar to verapamil. In Ca++ restricted Krebs solution, a rightward shift in the Ca++ response curves observed in the tracheal chain strips which were pretreated with Roflumilast (0.001–0.003 mg/mL) and the maximum response was suppressed, similarly as with verapamil. PDE inhibitory effect of Roflumilast evaluated by recording dose-dependent (0.03–0.1 mg/mL) responses, the isoprenaline-induced inhibitory dose response curves shifted leftward similar to papaverine (PDE inhibitor). Pretreatment with Roflumilast exhibited elevated intracellular cAMP levels in tracheal strips. Findings of the experiment conclude bronchodilatory influence of Roflumilast via PDE and Ca++ channel inhibition. Results of current experiment offers comprehensive mechanistic background of Roflumilast in future as therapeutic bronchodilator for hyperactive bronchial airway diseases.

Effect of Boswellia serrata on Rat Trachea Contractility In Vitro

Background: Boswellia serrata (family Burseraceae) has been traditionally used for the treatment of a wide variety of diseases as arthritis, inflammatory bowel diseases, and airway diseases. However, the direct bronchodilator efficacy of Boswellia serrata hasn’t been explored yet. Objective: We aimed at the present study to evaluate the direct effect of Boswellia serrata extract (BSE) on isolated rat tracheal preparations precontracted with either Acetylcholine (ACh) or potassium chloride (KCl). Methods: Tracheal rings were prepared from male Wistar rats (200-250 g). BSE (1-200 μg/ml) was added to tracheal strips precontracted with either ACh or KCl and the response was observed. We also investigated the consequences of epithelial denudation, indomethacin, and N-Nitro-L-arginine on the relaxant effect of BSE as compared to that of the β-adrenoceptor agonist isoprenaline, or the bitter taste receptor (TAS2R) agonist denatonium benzoate. Finally, the possible additive effects of BSE to isoprenaline or denatonium-induced relaxation were evaluated. Results: By using a set of serial dosing and washout experiments with tracheal rings, results showed that exposure to BSE resulted into a significant and concentration-dependent inhibitory effect on airway smooth muscle contractions precontracted with either ACh or KCl. Epithelial denudation, indomethacin, or N-Nitro-L-arginine had no significant effect on the obtained relaxation. Furthermore, BSE potentiated the relaxant effect of isoprenaline on rat trachea. Conclusion: BSE exerts a direct concentration-dependent relaxant effect on precontracted tracheal strips. These results could contribute towards validation of the traditional use of BSE in the treatment of airway diseases.

Potential effect of one-alpha-hydroxy vitamin D3 (alfacalcidol) on experimentally induced allergic asthma

Background: Alfacalcidol is a vitamin D analog mainly used in the management of osteoporosis. Apart from the well-known role of vitamin D in bone and calcium metabolism, an immune-modulator role of vitamin D in allergic diseases was suggested. Aim: To evaluate the potential effect of alfacalcidol either alone or in combination with salbutamol or/and prednisolone and its role in the protection and management of experimentally induced allergic asthma. Materials and methods: Male guinea pigs were divided into two groups: group A constituted normal guinea pigs (received saline) and group B constituted ovalbumin-sensitized guinea pigs, which were divided into eight subgroups: group B1 (control): groups from B2 to B4 was treated with salbutamol, prednisolone, alfacalcidol, respectively, for 1 week, while group B5 was treated with salbutamol+prednisolone; group B6 was treated with salbutamol+alfacalcidol; group B7 was treated with prednisolone+alfacalcidol; and group B8 was treated with salbutamol, prednisolone, and alfacalcidol for 1 week. Twenty-four hours after the last dose, the animals were subjected to both: (a)pharmacological studies, (b) histopathological studies (group A, group B1, B3, and B4). Results: (a) Pharmacological studies: sensitization of guinea pigs caused a significant increase in amplitude of histamine-induced contractions of isolated tracheal smooth muscles in comparison to that of the normal one. Treatment of sensitized guinea pigs with salbutamol, prednisolone, or alfacalcidol produced a significant reduction in the amplitude of histamine-induced contractions in comparison to sensitized nontreated guinea pigs. Addition of alfacalcidol to salbutamol or prednisolone in the treatment of sensitized guinea pigs caused more decrease in the amplitude of histamine-induced contractions. However, the highest reduction in the amplitude of contractions of isolated tracheal strips was produced by the treatment of guinea pigs with a combination of salbutamol, prednisolone, and alfacalcidol. (b) Histopathological examinations: treatment of sensitized guinea pigs with alfacalcidol resulted in an anti-remodeling effect, while prednisolone showed better anti-inflammatory effect, when it was compared with that of the control group. Conclusion: Alfacalcidol was shown to possess anti-inflammatory and anti-remodeling effects in allergic asthma. It was superior to prednisolone regarding the anti-remodeling effect. So, alfacalcidol could be beneficial in the management of allergic asthma.

Effect of Protease-Activated Receptor-2-Activating Peptide on Guinea Pig Airway Resistance and Isolated Tracheal Strips.

Purpose:Protease-activated receptors (PARs) are a family of G-protein-coupled receptors distributed in a number of tissues. PAR-2 is expressed on airway epithelium and smooth muscles and overexpressed under pathological conditions, such as asthma and chronic obstructive pulmonary disease. However, the role of PAR-2 in airways has not yet been defined. In this study, we investigated the role of PAR-2-activating peptide (SLIGRL) on histamine-induced bronchoconstriction and the mechanisms underlying the bronchoprotective effect both in vivo and in vitro.Materials and Methods:The effect of SLIGRL was tested in vivo using histamine-induced bronchoconstriction in the guinea pig and in vitro using isolated tracheal spiral strips.Results:In vivo pretreatment with SLIGRL significantly reduced the histamine-induced increased bronchoconstriction. Neither propranolol nor vagotomy abolished the inhibitory effect of SLIGRL. Furthermore, indomethacin or glibenclamide did not antagonize the inhibitory response to SLIGRL. In isolated tracheal spiral strips in vitro, SLIGRL did not affect the contractile response to acetylcholine or potassium chloride; however, histamine-induced contraction was inhibited in a dose-dependent manner.Conclusion:Our data demonstrate the protective effect of SLIGRL in airways; however, this effect appears to be mediated independently of prostanoids, nitric oxide, circulating adrenaline, ATP-sensitive K + channels, and vagal stimulation.

Shortening of airway smooth muscle is modulated by prolonging the time without simulated deep inspirations in ovine tracheal strips.

The shortening of airway smooth muscle (ASM) is greatly affected by time. This is because stimuli affecting ASM shortening, such as bronchoactive molecules or the strain inflicted by breathing maneuvers, not only alter quick biochemical processes regulating contraction but also slower processes that allow ASM to adapt to an ever-changing length. Little attention has been given to the effect of time on ASM shortening. The present study investigates the effect of changing the time interval between simulated deep inspirations (DIs) on ASM shortening and its responsiveness to simulated DIs. Excised tracheal strips from sheep were mounted in organ baths and either activated with methacholine or relaxed with isoproterenol. They were then subjected to simulated DIs by imposing swings in distending stress, emulating a transmural pressure from 5 to 30 cmH2O. The simulated DIs were intercalated by 2, 5, 10, or 30 min. In between simulated DIs, the distending stress was either fixed or oscillating to simulate tidal breathing. The results show that although shortening was increased by prolonging the interval between simulated DIs, the bronchodilator effect of simulated DIs (i.e., the elongation of the strip post- vs. pre-DI) was not affected, and the rate of re-shortening post-simulated DIs was decreased. As the frequency with which DIs are taken increases upon bronchoconstriction, our results may be relevant to typical alterations observed in asthma, such as an increased rate of re-narrowing post-DI.NEW & NOTEWORTHY The frequency with which patients with asthma take deep inspirations (DIs) increases during bronchoconstriction. This in vitro study investigated the effect of changing the time interval between simulated DIs on airway smooth muscle shortening. The results demonstrated that decreasing the interval between simulated DIs not only decreases shortening, which may be protective against excessive airway narrowing, but also increases the rate of re-shortening post-simulated DIs, which may contribute to the increased rate of re-narrowing post-DI observed in asthma.

Second M3 muscarinic receptor binding site contributes to bronchoprotection by tiotropium.

The bronchodilator tiotropium binds not only to its main binding site on the M3 muscarinic receptor but also to an allosteric site. Here, we have investigated the functional relevance of this allosteric binding and the potential contribution of this behaviour to interactions with long-acting β-adrenoceptor agonists, as combination therapy with anticholinergic agents and β-adrenoceptor agonists improves lung function in chronic obstructive pulmonary disease. ACh, tiotropium, and atropine binding to M3 receptors were modelled using molecular dynamics simulations. Contractions of bovine and human tracheal smooth muscle strips were studied. Molecular dynamics simulation revealed extracellular vestibule binding of tiotropium, and not atropine, to M3 receptors as a secondary low affinity binding site, preventing ACh entry into the orthosteric binding pocket. This resulted in a low (allosteric binding) and high (orthosteric binding) functional affinity of tiotropium in protecting against methacholine-induced contractions of airway smooth muscle, which was not observed for atropine and glycopyrrolate. Moreover, antagonism by tiotropium was insurmountable in nature. This behaviour facilitated functional interactions of tiotropium with the β-agonist olodaterol, which synergistically enhanced bronchoprotective effects of tiotropium. This was not seen for glycopyrrolate and olodaterol or indacaterol but was mimicked by the interaction of tiotropium and forskolin, indicating no direct β-adrenoceptor-M3 receptor crosstalk in this effect. We propose that tiotropium has two binding sites at the M3 receptor that prevent ACh action, which, together with slow dissociation kinetics, may contribute to insurmountable antagonism and enhanced functional interactions with β-adrenoceptor agonists.

Pharmacological Studies Pertaining to Smooth Muscle Relaxant, Platelet Aggregation Inhibitory and Hypotensive Effects of Ailanthus altissima.

Objective This in vitro and in vivo study was conducted to rationalize some of traditional medicinal uses of Ailanthus altissima in gastrointestinal, respiratory, and cardiovascular systems. Materials Crude extract of Ailanthus altissima (Aa.Cr) and its fractions were prepared and utilized in in vitro and in vivo studies. For in vitro studies, Aa.Cr was investigated on isolated rabbit jejunum, isolated tracheal strip, and isolated aorta of rat suspended in tissue organ bath. Platelet rich and platelet poor plasma were used to study platelet aggregation inhibitory activity. In vivo antidiarrheal effect of Aa.Cr was investigated on balb/c mice pretreated with castor oil to induce diarrhea and SD rats were used to study hypotensive activity. Results Concentration dependent spasmolytic effects of Aa.Cr and its DCM fraction (Aa.DCM) were observed on spontaneous and spasmogen induced contractions in jejunum isolated from rabbit, but effect against high potassium (high-K+) induced contractions was more potent. Moreover Aa.Cr showed parallel shifting of calcium response curve to the right side. While its aqueous fraction (Aa.aq) caused spasmogenesis of isolated rabbit jejunum, this effect was blocked partially with prior administration of atropine (1μM). Concentration dependent protection against castor oil induced diarrhea was also observed. Relaxant effect was observed by the application of Aa.Cr and Aa.DCM against high-K+ and carbachol (CCh) induced contractions in tracheal strips isolated from SD rats, while Aa.Aq caused partial relaxation of high-K+ induced contractions, but no effect was observed against CCh induced contractions. Relaxation of rat aorta by the application of Aa.Cr and its fractions was also observed. Inhibition of force of contraction in rabbit atrium was also observed. Inhibition of platelet aggregation was observed against epinephrine and ADP induced aggregation. Conclusion Keeping in view the observed results, it is concluded that smooth muscle relaxant, platelet aggregation inhibitory and hypotensive effect may be due to the blockage of calcium channels.

Mechanopharmacology and Synergistic Relaxation of Airway Smooth Muscle.

Asthmatic airways are stiffer than normal. We have shown that the cytoskeletal passive stiffness of airway smooth muscle (ASM) can be regulated by intracellular signaling pathways, especially those associated with Rho kinase (ROCK). We have also shown that an oscillatory strain reduces the passive stiffness of ASM and its ability to generate force. Here, we investigated the combined effect of inhibiting the ASM contraction with β2 agonist and decreasing the ASM cytoskeletal stiffness with ROCK inhibitor and/or force oscillation (FO) on the relaxation of contracted ASM. We hypothesize that the ASM relaxation can be synergistically enhanced by the combination of these interventions, because drug-induced softening of the cytoskeleton enhances the FO-induced relaxation and vice versa. Sheep tracheal strips were isotonically contracted to acetylcholine (3 × 10−5 M). At the plateau of shortening, β2 agonist salbutamol (10−7 M), ROCK inhibitor H1152 (10−7 M), and FO (square wave, 1 Hz, amplitude 6% maximal active force) were applied either alone or in combination. After adjusting for nonspecific time-dependent variation, relengthening by individual interventions with low-dose salbutamol or H1152, or small amplitude FO was not significantly different from zero. However, significant relengthening was observed in all combination treatments. The relengthening was greater than the mathematical sum of relengthening caused by individual treatments thereby demonstrating synergistic relaxation. The ASM stiffness did not change with salbutamol or H1152 treatments, but was lower with FO in combination with H1152. The results suggest that the mechanopharmacological treatment can be an effective therapy for asthma.

Attenuation of murine and human airway contraction by a peptide fragment of the cytoskeleton regulatory protein gelsolin.

We have previously reported that mice genetically deficient in the actin binding protein gelsolin exhibit impaired airway smooth muscle (ASM) relaxation. Primary cultured ASM cells from these mice demonstrate enhanced inositol triphosphate (IP3) synthesis and increased intracellular calcium in response to Gq-coupled agonists. We hypothesized that this was due to increased intracellular availability of unbound phosphatidylinositol 4,5-bisphosphate (PIP2), based on the fact that gelsolin contains a short peptide region that binds PIP2, presumably making it a less available substrate. We now questioned whether a peptide that corresponds to the PIP2 binding region of gelsolin could modulate ASM signaling and contraction. The 10 amino acid sequence of the gelsolin peptide within the PIP2-binding region was incubated with primary cultures of human ASM cells, and IP3 synthesis was measured in response to a Gq-coupled agonist. Gelsolin peptide-treated cells generated less IP3 under basal and bradykinin or acetylcholine (Gq-coupled) conditions. Acetylcholine-induced contractile force measured in isolated tracheal rings from mice and human tracheal muscle strips in organ baths was attenuated in the presence of the gelsolin peptide. The gelsolin peptide also attenuated methacholine-induced airway constriction in murine precision-cut lung slices. Furthermore, this peptide fragment delivered to the respiratory system of mice via nebulization attenuated subsequent methacholine-induced increases in airway resistance in vivo. The current study demonstrates that introduction of this small gelsolin peptide into the airway may be a novel therapeutic option in bronchoconstrictive diseases.

Heating-induced contraction in airways smooth muscle: A possible causative factor of exercise-induced bronchoconstriction

Asthmatic patients often suffer from bronchoconstriction or asthma following breathing hot air or exposure to exercise due to raises the core body temperature. However, the direct effect of heating airways has not been studied yet. The aim of this study is to investigate the effect of heating on tracheal and bronchiolar smooth muscles. Isolated ovine tracheal strips and bronchiolar segments preparations were suspended in organ baths containing Krebs’ solution for isometric tension recording. Tissues responses were examined during decreasing and elevating baths temperature (20 °C, 40 °C–45 °C and 50 °C). Cooling or heating induced rapid and reproducible contractions proportional with decreasing or increasing temperature respectively in tracheal and bronchiolar preparations. On reset to 37 °C the tone returned rapidly to basal level. Changing the bath’s temperature from 37 °C to 20 °C or to 40 °C and 45 °C for tracheal strips or to 45 °C and 50 °C for bronchiolar segments induced contractions in both preparations. Changing the temperature below or above the normal body temperature (37 °C), leads to airways contractions. Heating induced contractions in tracheal and bronchiolar smooth muscle proportional to the heating temperature. Therefore, breathing hot air or elevation of body core temperature due to exercise can be considered possible causative factor of heating- or exercise-induced bronchoconstriction.

INHIBITORY EFFECT OF ETHANOLIC EXTRACT OF ERIOBOTRYA JAPONICA LEAVES PRE-INCUBATED WITH THEOPHYLLINE AND ASPIRIN ON ISOLATED GUINEA PIG TRACHEAL

Objectives: The objectives of the study were to examine the inhibitory effect of ethanol extract of Eriobotrya japonica leaves (EEEJL) pre-incubated with theophylline and aspirin on isolated guinea pig tracheal chains against acetylcholine (ACh)-induced contraction.Methods: The effect of EEEJL (1-8 mg/Ml) on the isolated tracheal strips was tested in vitro. Furthermore, the mechanism of relaxant effects of EEEJL was evaluated in the presence of theophylline and aspirin.Results: The contractile response evoked by Ach (1.25 × 10−3 M) was decreased by EEEJL (effective concentration50 = 1.36 mg/mL) and has no significant difference of relaxant effect to that of EEEJL pre-incubated with theophylline and aspirin (p&gt;0.05).Conclusion: The EEEJL decreased the ACh-induced contraction through the inhibition of PDE and the protective effect on prostaglandin E2.

Modulation of human airway smooth muscle biology by human adipocytes

BackgroundAsthma and obesity, two growing epidemics worldwide, may share an underlying causal relationship. Airway hyperresponsiveness (AHR), a defining component of asthma, has been documented in both ‘obese’ animal models and non-asthmatic obese individuals. However, there is a paucity of evidence that obesity-derived factors directly affect human airway smooth muscles (ASM).MethodsExperiments were designed with primary ASM and adipocytes isolated from the same human tissue explants (n = 6). The modulatory effects of human adipocytes extracted from subcutaneous (extrathoracic) and visceral (intrathoracic) depots, on ASM biology was examined with respect to proliferation, migration, contractility and pro-inflammatory cytokine synthesis.ResultsAdipocyte-conditioned media as well as myocyte-adipocyte co-cultures failed to show any significant changes in the proliferative or migrational properties of the ASM. Adipocyte-conditioned media also had no effect on the contractility or relaxation of bovine tracheal muscle strips. In contrast, there was a moderate yet significant increase of IL-6 and eotaxin release by ASM incubated with adipocyte-conditioned media (P = 0.0035 and P = 0.0067, vs. control, respectively), thereby further consolidating the altered inflammatory state reported for both diseases.ConclusionWe report, for the first time, that adipocytes from either subcutaneous or visceral depots can trigger an inflammatory state in the ASM, with negligible modulatory effects on hyperplasia, hypertrophy or contractile properties.

Enhanced basal tension in isolated rat tracheal smooth muscle stimulated by electric field stimulation under low temperature.

Exposure to cold causes cutaneous vasoconstriction to reduce body heat loss, while the airway warms up the inspired cold air, thus suggesting that cooling might evoke a response in tracheal smooth muscle different from that in cutaneous blood vessels. The aim of this study was to evaluate the effect of temperature on isolated rat trachea, with or without electric field stimulation (EFS). Tissue bath for isolated trachea was used. An in vitro isometric contraction of trachea from healthy male Sprague-Dawley rat (body weight: ≥ 200 g) was continuously recorded. Tension in strips of rat trachea that were untreated and treated with EFS, was continuously recorded in stepwise manner at temperatures varying from 37 °C to 7 °C or from 7 °C to 37 °C. Results indicated that descent and re-ascent of temperature produced temperature-dependent tension changes. Basal tension of the trachea decreased when temperature was reduced if EFS was not applied. EFS-induced spike contraction decreased when temperature was reduced, while basal tension increased at the same time. We concluded that low temperature induced rapid and reproducible contraction in isolated rat tracheal strip only if EFS was applied. Increasing temperature reduced basal tension and enhanced EFS-induced spike contraction of the trachea at the same time.