Tracheal Contraction Research Articles

The T-Type Calcium Channel CACNA1H is Required for Smooth Muscle Cytoskeletal Organization During Tracheal Tubulogenesis.

Abnormalities of tracheal smooth muscle (SM) formation are associated with several clinical disorders including tracheal stenosis and tracheomalacia. However, the cellular and molecular mechanisms underlying tracheal SM formation remain poorly understood. Here, it is shown that the T-type calcium channel CACNA1H is a novel regulator of tracheal SM formation and contraction. Cacna1h in an ethylnitrosourea forward genetic screen for regulators of respiratory disease using the mouse as a model is identified. Cacna1h mutants exhibit tracheal stenosis, disorganized SM and compromised tracheal contraction. CACNA1H is essential to maintain actin polymerization, which is required for tracheal SM organization and tube formation. This process appears to be partially mediated through activation of the actin regulator RhoA, as pharmacological increase of RhoA activity ameliorates the Cacna1h-mutant trachea phenotypes. Analysis of human tracheal tissues indicates that a decrease in CACNA1H protein levels is associated with congenital tracheostenosis. These results provide insight into the role for the T-type calcium channel in cytoskeletal organization and SM formation during tracheal tube formation and suggest novel targets for congenital tracheostenosis intervention.

Chamaecyparis lawsoniana and Its Active Compound Quercetin as Ca2+ Inhibitors in the Contraction of Airway Smooth Muscle.

The Cupressaceae family includes species considered to be medicinal. Their essential oil is used for headaches, colds, cough, and bronchitis. Cedar trees like Chamaecyparis lawsoniana (C. lawsoniana) are commonly found in urban areas. We investigated whether C. lawsoniana exerts some of its effects by modifying airway smooth muscle (ASM) contractility. The leaves of C. lawsoniana (363 g) were pulverized mechanically, and extracts were obtained by successive maceration 1:10 (w:w) with methanol/CHCl3. Guinea pig tracheal rings were contracted with KCl, tetraethylammonium (TEA), histamine (HIS), or carbachol (Cch) in organ baths. In the Cch experiments, tissues were pre-incubated with D-600, an antagonist of L-type voltage-dependent Ca2+ channels (L-VDCC) before the addition of C. lawsoniana. Interestingly, at different concentrations, C. lawsoniana diminished the tracheal contractions induced by KCl, TEA, HIS, and Cch. In ASM cells, C. lawsoniana significantly diminished L-type Ca2+ currents. ASM cells stimulated with Cch produced a transient Ca2+ peak followed by a sustained plateau maintained by L-VDCC and store-operated Ca2+ channels (SOCC). C. lawsoniana almost abolished this last response. These results show that C. lawsoniana, and its active metabolite quercetin, relax the ASM by inhibiting the L-VDCC and SOCC; further studies must be performed to obtain the complete set of metabolites of the extract and study at length their pharmacological properties.

The Bronchodilator Potential of Astragalus sarcocolla: An in vitro Experiment.

To evaluate the bronchodilatory mechanism of Astragalus sarcocolla (ASE) extract on tracheal smooth muscles of rabbits. In-vitro experimental study. Place and Duration of the Study: The animal house of CMH Lahore Medical College, Lahore, and Institute of Dentistry, NUMS, from October 2022 to May 2023. Six rabbits were randomly divided into four groups. After euthanising the rabbit, the trachea was carefully dissected out and stabilised in Kreb's Henseleit solution for 30 minutes and then, stimulated by acetylcholine (Ach) 1µm, under mimicked physiological conditions. Group I served as the control group with tracheal smooth muscles stabilised with 1g tension. In Group II (positive control), tracheal smooth muscles were stimulated by potassium chloride (KCl) (80 mM and 25 mM, respectively) to get maximum tracheal smooth muscle contractions. Later, the tissue was exposed to theophylline with three molar concentrations 0.2, 0.4, 0.6, and 0.8 mM, and cumulative dose response curves were formed. In Group III (ASE group), tracheal smooth muscles were stimulated by KCl (80 mM and 25 mM) and was exposed to increasing concentration of ASE. In group IV, tissue was stimulated by KCl (25 mM) and glibenclamide (3 µM), later exposed to increasing concentration of ASE to confirm the bronchodilatory mechanism. The change in isometric contraction of the tissue was recorded using the force displacement transducer connected to a PowerLab data acquisition system. Concentration response curves were drawn, and median effective concentrations (EC50 values) and percentage inhibition were calculated. Non-linear regression was applied for the analysis of the concentration-response curves. ASE inhibited the KCl-induced low potassium (25 mM) contractions (EC50 = 0.38 mg/ml, 95% CI: 0.04 - 0.38, n = 6). It only partially inhibited the high potassium-induced contractions in tracheal smooth muscles. Pretreatment with glibenclamide showed a rightward shift of the dose-response curve. Theophylline and ASE significantly reduced the low K+ induced smooth muscle contractions in comparison to the control group (p <0.001, each). Astragalus sarcocolla extract produced bronchodilator effects through the activation of ATP sensitive potassium channels in isolated rabbit trachea. Astragalus sarcocolla, Bronchodilators, ATP-sensitive potassium channels, Effective concentration 50, Concentration response curves.

Desmopressin nasal spray inhibiting parasympathetic function on isolated tracheal smooth muscle.

Objectives: Nocturia with or without asthma is one of the aging diseases. Desmopressin has been used as a nasal spray for patients who are suffering from nocturia. This study determined the effects of desmopressin on isolated tracheal smooth muscle in vitro. Methods: We evaluated desmopressin's efficiency on isolated rat tracheal smooth muscle. Desmopressin was evaluated for the following effects on tracheal smooth muscle: (1) effect on resting tension; (2) effect on contraction brought on by parasympathetic mimetic 10-6 M methacholine; and (3) effect on electrically produced tracheal smooth muscle contractions. Results: As the concentration grew, desmopressin by itself had no impact on the trachea's baseline tension. Addition of desmopressin at doses of 10-5 M or above elicited a significant relaxation response to 10-6 M methacholine-induced contraction. Desmopressin could also inhibit spike contraction of the trachea induced by electrical field. Conclusion: According to this study, desmopressin at high quantities may prevent the trachea's parasympathetic activity. Due to its ability to block parasympathetic activity and lessen the contraction of the tracheal smooth muscle brought on by methacholine, Desmopressin nasal spray might help nocturia sufferers experience fewer asthma attacks.

An In Vitro and In Silico Characterization of Salvia sclarea L. Methanolic Extracts as Spasmolytic Agents.

The use of medicinal plant species and their products is widespread in the field of gastrointestinal and respiratory diseases. This study aimed to evaluate the traditional use of Salvia sclarea L., clary sage, finding the possible mechanisms of its spasmolytic and bronchodilator actions in in vitro conditions supported by molecular docking analysis, along with the antimicrobial effects. Four dry extracts were prepared from the aerial parts of S. sclarea, using absolute or 80% (v/v) methanol by the method of a single-stage maceration or an ultrasound-assisted extraction. Characterization of the bioactive compounds by high-performance liquid chromatography indicated the presence of significant amounts of polyphenolics, with rosmarinic acid as the prevalent one. The spontaneous ileal contractions were best inhibited by the extract prepared with 80% methanol and maceration. The same extract was superior in the carbachol- and KCl-induced tracheal smooth muscle contractions, being the strongest bronchodilator agent. The most powerful relaxation of KCl-induced ileal contractions was achieved with the extract made of absolute methanol by maceration, while the 80% methanolic extract made with the ultrasound method generated the best spasmolytic effects in the acetylcholine-induced ileal contractions. Docking analysis suggested that apigenin-7-O-glucoside and luteolin-7-O-glucoside exhibited the highest binding affinity to voltage-gated calcium channels. Gram (+) bacteria were more susceptible to the effects of the extracts, particularly Staphylococcus aureus, in contrast to Gram (-) bacteria and Candida albicans. This is the first study to point out the influence of S. sclarea methanolic extracts on the gastrointestinal and respiratory spasm reduction, paving the way for their potential place in complementary medicine.

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.

The benefits of Salvia sclarea L. ethanolic extracts on gastrointestinal and respiratory spasms

Aromatic herbal drugs have been widely used in the treatment of many respiratory and gastrointestinal diseases as part of traditional medicine. Many recent studies have shown that plant species of the Lamiaceae family and their products express relaxant effects on the contractions of the isolated ileal and tracheal smooth muscle of experimental animals. There is no study reporting the spasmolytic effects of the Salvia sclarea L. (clary sage, family Lamiaceae) extracts, therefore, the aim of the study was to examine their effects on spontaneous, KCl-, acethylcholine-, histamine-, BaCl2- and CaCl2- induced rats ileal contractions, and carbachol- and KCl- induced tracheal smooth muscle contractions. Two extracts were prepared from the aerial parts of the herb using concentrate (96%) or 80% ethanol in an ultrasonic bath. In addition to determining the spasmolytic effect of the extracts, the chemical composition and antimicrobial effects against six Gram (+) and six Gram (-) bacteria and one yeast (microdilution assay) were also investigated. The extracts were rich in polyphenolics wherein rosmarinic acid was the predominant compound. Higher concentrations of phenolic acids and flavonoid heterosides were found in the extract prepared with 80% ethanol while flavonoid aglycones were more represented in the extract prepared with concentrated ethanol. The S. sclarea extracts significantly inhibited the rats ileum and tracheal contractions in a concentration dependent regime. The extract prepared with concentrated ethanol was a better relaxant agent of ileum smooth muscles in spontaneous, KCl- and CaCl2- induced contractions, and the extract prepared with 80% ethanol was superior in other series of experiments. Gram (+) bacteria, especially Staphylococcus aureus, were more sensitive to the action of the S. sclarea extracts in comparison to Gram (-). The extracts were not effective in the anticandidal part of the microdilution assay. Based on these results, S. sclarea could find an appropriate place in modern phytotherapy as a potential treatment agent of gastrointestinal and respiratory diseases associated with spasms.

Docosahexaenoic Acid Selectively Suppresses U46619- and PGF2α-Induced Contractions in Guinea Pig Tracheal Smooth Muscles.

We investigated the potential inhibitory effects of docosahexaenoic acid (DHA) on the contractions of guinea pig tracheal smooth muscles in response to U46619 (a thromboxane A2 (TXA2) mimetic) and prostaglandin F2α (PGF2α) to examine whether this n-3 polyunsaturated fatty acid suppresses prostanoid-induced tracheal contractions. DHA (3 × 10-5 M) significantly suppressed tracheal contractions elicited by lower concentrations of U46619 (10-8 M) and PGF2α (5 × 10-7 M) (vs. control), although it did not suppress the contractions induced by higher concentrations (U46619: 10-7 M; PGF2α: 10-5 M). Supporting these findings, DHA (4 × 10-5 M/6 × 10-5 M) shifted the concentration-response curves for U46619 (10-9-10-6 M) and PGF2α (10-8-10-5 M) to the right. However, the slope of the regression line in the Schild plot of DHA vs. U46619/PGF2α was larger than unity. The tracheal contractions induced by U46619 (10-8 M) and PGF2α (5 × 10-7 M) were significantly suppressed by the prostanoid TP receptor antagonist SQ 29,548 (10-6 M) (vs. ethanol-treated). In contrast, DHA (4 × 10-5 M) did not show significant inhibitory effects on the contractions induced by acetylcholine (10-8-10-4 M), histamine (10-8-10-4 M), and leukotriene D4 (10-11-10-7 M) (vs. ethanol-treated). These findings indicate that DHA selectively suppresses tracheal contractions induced by U46619 and PGF2α. Therefore, DHA may be a useful therapeutic agent against asthma associated with tracheal/bronchial hyper-constriction caused by prostanoids including TXA2 and PGF2α.

International Conference on Recent updates in Disease Treatments - EYAN 2021

This special issue is highly related to Microscopic examinations of the tissue of the medulla from the corticalsubcortical region on the injured side after a traumatic brain injury and various exercises were performed. Also it focuses on to identify possible ways to protect the rights of people who have lost their jobs due to the spread of COVID-19, analyse the effectiveness of international experience and propose promising changes to labour legislation in this area. In addition, grotechnical Methods of Increasing Drought Resistance of Spring Barley, The Role of Il4 in Contraction of Tracheal Muscle Among Rats, Stabilization of Mast Cell Membranes by Sodium Cromoglicate in Smooth Muscle Contraction of Trachea and Bronchi of The Rat, are been focused in this issues. This special issue focuses on those spects with much implication for the health care. The articles published in this special issue will certainly bring as positive effect for the developing health care and to make use of available resources and to remove certain obsolete factors and process which may delay or harm the existing health care system. It enhances maximum utilization of scientific knowledge to potentiate therapy and diagnosis in the health care system. With the above discussion in mind, the papers are broadly divided into those describing applications of biotechnology and nanotechnology in biological sense. This Special Issue presents range of applications for biotechnology and nanobiotechnology for human health and environment. It is hoped that the reader will gain, from a reading of these papers a better appreciation and recent advances in the area.

Study of the expression of receptors to IL4, IL4R genes in trachea and the role of IL4 in contraction of tracheal muscle among rats

Study of the expression of receptors to IL4, IL4R genes in trachea and the role of IL4 in contraction of tracheal muscle among rats

Cellular Mechanism Underlying the Facilitation of Contractile Response Induced by Tumor Necrosis Factor-α in Mouse Tracheal Smooth Muscle

The proinflammatory cytokine tumor necrosis factor-α (TNF-α) augments intracellular Ca2+ signaling and contractile responses of airway smooth muscles, leading to airway hyperresponsiveness. However, the underlying mechanism has not been fully elucidated. This study aimed to investigate the cellular mechanism of the potentiated contraction of mouse tracheal smooth muscle induced by TNF-α. The results showed that TNF-α triggered facilitation of mouse tracheal smooth muscle contraction in an epithelium-independent manner. The TNF-α-induced hypercontractility could be suppressed by the protein kinase C inhibitor GF109203X, the tyrosine kinase inhibitor genistein, the Src inhibitor PP2, or the L-type voltage-dependent Ca2+ channel blocker nifedipine. Following TNF-α incubation, the α1C L-type Ca2+ channel (CaV1.2) was up-regulated in cultured primary mouse tracheal smooth muscle cells. Pronounced phosphotyrosine levels were observed in mouse tracheas. In conclusion, this study shows that TNF-α enhanced airway smooth muscle contraction via protein kinase C-Src-CaV1.2 pathways, which provides novel insights into the pathologic role of proinflammatory cytokines in mediating airway hyperresponsiveness.

The Spasmolytic, Bronchodilator, and Vasodilator Activities of Parmotrema perlatum Are Explained by Anti-Muscarinic and Calcium Antagonistic Mechanisms.

Parmotremaperlatum is traditionally used in different areas of Pakistan to treat gastrointestinal, respiratory, and vascular diseases. This study evaluates the underlying mechanisms for traditional uses of P. perlatum in diarrhea, asthma, and hypertension. In vitro pharmacological studies were conducted using isolated jejunum, trachea, and aortic preparations, while the cytotoxic study was conducted in mice. Crude extract of P. perlatum(Pp.Cr), comprising appreciable quantities of alkaloids and flavonoids, relaxed spontaneously contracting jejunum preparation, K+ (80 mM)-induced, and carbachol (1 µM)-induced jejunum contractions in a concentration-dependent manner similar to dicyclomine and dantrolene. Pp.Cr showed a rightward parallel shift of concentration-response curves (CRCs) of Cch after a non-parallel shift similarto dicyclomine and shifted CRCs of Ca+2 to rightward much likeverapamil and dantrolene, demonstrating the coexistence of antimuscarinic and Ca+2 antagonistic mechanism. Furthermore, Pp.Cr, dicyclomine, and dantrolene relaxed K+ (80 mM)-induced and Cch (1 µM)-induced tracheal contractions and shifted rightward CRCs of Cch similar to dicyclomine, signifying the dual blockade. Additionally, Pp.Cr also relaxed the K+ (80 mM)-induced and phenylephrine (1 µM)-induced aortic contraction, similarly to verapamil and dantrolene, suggesting Ca+2 channel antagonism. Here, we explored for the first time thespasmolytic and bronchodilator effects of Pp.Crand whether they maybe due to the dual blockade of Ca+2 channels and muscarinic receptors, while the vasodilator effect might be owing to Ca+2 antagonism. Our results provide the pharmacological evidence that P. perlatum could be a new potential therapeutic option to treat gastrointestinal, respiratory, and vascular diseases. Hence, there is a need for further research to explore bioactive constituent of P. perlatum as well as further investigation by suitable experimental models are required to further confirm the importance and usefulness of P. perlatum in diarrhea, asthma, and hypertension treatment.

Effect of TNF-α, IL-2, IL-5 and IL-6 on Rat Tracheal and Bronchial Smooth Muscle Contractions

Effect of TNF-α, IL-2, IL-5 and IL-6 on Rat Tracheal and Bronchial Smooth Muscle Contractions

Elucidation of Mechanisms in Cu (II) Caused Hypercontraction of Rat Tracheal Rings.

Airway smooth muscle contraction is one of the primary factors involved in the initiation and progression of asthma which in turn is regulated by increased cytosolic Ca2+ concentration from intracellular stores and through transmembrane ion channels. Calcium-independent factors such as reactive oxygen species (ROS) generation, nitric oxide (NO) depletion and cyclooxygenase (COX) pathways also contribute to tracheal smooth muscle contraction. Studies on copper toxicity suggest significance of this essential micronutrient overdose in acute respiratory disorders, allergic asthma and ciliary motion in tracheal explants. However, the mechanism of copper caused hypercontraction upon direct exposure to tracheal smooth muscle is largely unknown. In this study we investigate the effect of copper exposure on isolated tracheal rings and relative contributions of various factors in acetylcholine-induced contractions. Results obtained suggest that rise in intracellular calcium concentration via voltage-operated Ca2+ channel (VOCC), store-operated Ca2+ channel (SOCC), stretch-activated channels (SAC) and TRP channel (transient receptor potential channel) activation is the major factor in copper-mediated hypercontraction. ROS generation or COX-dependent pathways do not appear to significantly contribute to Cu2+ caused hypercontraction.

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.

Aster tataricus attenuates asthma efficiently by simultaneously inhibiting tracheal ring contraction and inflammation

Asthma is an airway chronic inflammatory disease with significant morbidity, mortality and huge social economic burden. Previous research demonstrated that the root of Aster tataricus (RA) may have the potential to treat asthma, but the efficacy and mechanism were not clear. In this study, preliminary results in vitro showed that Fr-75 eluted from RA extract could not only completely inhibit the tracheal ring contraction raised by KCl in 20 min, but also effectively affect the tracheal ring contraction induced by KCl-, Ach- and His in a concentration-dependent manner (3.91−250 μg/mL). Further results on cells exhibited that Fr-75 could decrease the concentration of intracellular Ca2+ as well. These results revealed the underlying mechanism in vitro that the inhibition of tracheal ring contraction might be due to the decline of the intracellular Ca2+ concentration, which caused by suppressing calcium channel, antagonizing the muscarinic and histamine receptors. Also, results in vivo exhibited that Fr-75 could distinctly ease the symptoms of ovalbumin-sensitized mice, including relieving the pathological injury, increasing the latency to preconvulsive dyspnea and to enhanced pause, reducing the inflammatory cells, chemokines and cytokines in BALF and lung tissue. In general, it could be speculated that RA fraction may attenuate asthma through dilating the tracheal ring contraction and alleviating the lung inflammation simultaneously.

Anti-Bronchospasmodic Effect of JME-173, a Novel Mexiletine Analog Endowed With Highly Attenuated Anesthetic Activity.

Local anesthetics (LAs), such as lidocaine and mexiletine, inhibit bronchoconstriction in asthmatics, but adverse effects limit their use for this specific clinical application. In this study, we describe the anti-spasmodic properties of the mexiletine analog 2-(2-aminopropoxy)-3,5-dimethyl, 4-Br-benzene (JME-173), which was synthesized and screened for inducing reduced activity on Na+ channels. The effectiveness of JME-173 was assessed using rat tracheal rings, a GH3 cell line and mouse cardiomyocytes to access changes in smooth muscle contraction, and Na+, and Ca++ionic currents, respectively. Bronchospasm and airway hyper-reactivity (AHR) were studied using whole-body barometric plethysmography in A/J mice. We observed that the potency of JME-173 was 653-fold lower than mexiletine in inhibiting Na+ currents, but 12-fold higher in inhibiting L-type Ca++ currents. JME-173 was also more potent than mexiletine in inhibiting tracheal contraction by carbachol, allergen, extracellular Ca++, or sodium orthovanadate provocations. The effect of JME-173 on carbachol-induced tracheal contraction remained unaltered under conditions of de-epithelized rings, β2-receptor blockade or adenylate cyclase inhibition. When orally administered, JME-173 and theophylline inhibited methacholine-induced bronchospasm at time points of 1 and 3 h post-treatment, while only JME-173 remained active for at least 6 h. In addition, JME-173 also inhibited AHR in a mouse model of lipopolysaccharide (LPS)-induced lung inflammation. Thus, the mexiletine analog JME-173 shows highly attenuated activity on Na+ channels and optimized anti-spasmodic properties, in a mechanism that is at least in part mediated by regulation of Ca++ inflow toward the cytosol. Thus, JME-173 is a promising alternative for the treatment of clinical conditions marked by life-threatening bronchoconstriction.

Ex‐vivo Effects of Limonene in Airways and Vasculature in Allergic Adenosine A2A receptor Knock‐out and Wild‐type mice

Limonene, a mono‐terpene flavonoid, has anti‐inflammatory effects in asthma by activating A2A adenosine receptors. We studied the effects of inhaled d‐limonene on isolated aortic and tracheal rings from asthmatic mice using tissue/organ bath system. We used mice in which the A2A receptor was genetically knocked out (A2AKO) and C57BLJ/6 (wild‐type; WT) for the study. The mice were divided into control (CON) and allergen‐induced sensitized (SEN), control mice treated with limonene through aerosol route (CON+LIM) and allergen‐induced sensitized mice treated with limonene (SEN+LIM). Mice were sensitized i.p. on days 1, 6 with 20μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. Control groups were given an i.p injection of vehicle (1,6 days) and the aerosol challenge of normal saline (11–13 days). SEN +LIM and CON +LIM groups were given 0.01% limonene in the form of aerosol 90 min prior to the OVA and saline challenge on days 11,12,13. Trachea and aorta were isolated on day 14 for further experiments. Tissue responses were studied in the form of contraction and relaxations in all groups. Treatment with limonene significantly reduced the tracheal reactivity (80.68±20% in SEN+LIM vs SEN; p&lt;0.05) to methacholine (MCh) in WT mice that were allergic. MCh induced tracheal contractions were not altered in A2AKO limonene treated sensitized group compared to non‐treated A2AKO sensitized group (209.87 ± 41.33 A2AKO SEN+LIMSEN+LIM Vs 212.51 ± 43.18 in A2AKO SEN) In presence of A2A‐antagonist (SCH58651), a significant increase in the MCh induced contractility in WT SEN was observed(341.65 ± 2.664 SEN+SCH Vs 225.99 ± 67.34 SEN, pP&lt;0.05). A2A agonist CGS 21680 induced relaxation in WT CON was abolished in WT SEN but restored in WT SEN+LIM (37.8± 16.4% Vs 1.2± 11.1 in SEN, p&lt;0.05). The CGS‐induced tracheal relaxation in WT SEN+LIM and SEN groups was increased in the presence of the NADPH inhibitor apocynin (60.58 ± 17.20 SEN; 21.28 ± 16.02 SEN). In the aorta, acetylcholine‐induced aortic relaxation (endothelium‐dependent response) in WT SEN group was significantly lower than controls (29.82 ± 5.62 WT SEN Vs 51.51 ± 30.13 WT CON, p&lt;0.05) with limonene restoring the response (61.56 ± 11.34 WT‐SEN+LIM). CGS 21680 induced aortic relaxation in WT SEN was completely blunted compared to WT CON (5.62±3.72 WT SEN Vs 66.91 ± 6.8 WT‐CON, *p&lt;0.05). Treatment with limonene restored CGS 21680‐induced relaxation in asthmatic mice (63.8 ± 6.32 WT SEN+LIM Vs 5.62± 3.72 SEN) In conclusion, it appears that limonene restores the tracheal and aortic relaxation induced by adenosine‐mediated pathways by activating the A2A receptor in asthmatic mice.Support or Funding InformationSupported by LIU start‐up funds (DSP), HL027339 (SJM)

Eucalyptol reduces airway hyperresponsiveness in rats following cigarette smoke-exposed

BackgroundCigarette smoke is the major cause of airway inflammatory disease, including airway hyperresponsiveness. Eucalyptol (EUC), also named 1.8-cineole, is a monoterpenoid found in essential oil of medicinal plants, showing several biological effects. Hypothesis/purposeBased in the eucalyptol protective activity in respiratory diseases as asthma, our hypothesis is that eucalyptol is able to reduce the airway hyperresponsiveness and the respiratory mechanic parameters in rats exposed to cigarette smoke. Study designWistar rats were divided into control and cigarettes smoke (CS) groups. CS group was daily subjected to cigarette smoke and treated by inhalation for 15 min/day with EUC (1 mg/mL) or vehicle during 30 days. After treatment, bronchoalveolar lavage (BAL) was collected to analyze the inflammatory profile, and tracheal rings were isolated for evaluation of the airway smooth muscle hyperresponsiveness. Lung function was analyzed in vivo. MethodsThe inflammatory profile was evaluated by optical microscopy performing total (Neubauer chamber) and differential leukocyte count (smear slides stained in H&E). The hyperresponsiveness was evaluated in tracheal rings contracted with potassium chloride (KCl) carbamoylcholine (CCh), or Barium chloride (BaCl2) in presence or absence of nifedipine. The lung function (Newtonian resistance-RN) was evaluated by bronco stimulation with methacholine (MCh). ResultsBAL from CS group increased the influx of leukocyte, mainly neutrophils and macrophages compared to control group. EUC reduced by 71% this influx. The tracheal contractions induced by KCl, CCh or BaCl2 were reduced by EUC in 59%, 42% and 26%, respectively. The last one was not different of nifedipine activity. Newtonian resistance (RN) was also reduced in 37% by EUC compared to CS group. ConclusionEUC reduces the hyperresponsiveness and the airway inflammatory profile, recovering the lung function.

Bronchodilator effect of apigenin and luteolin, two components of Dracocephalum kotschyi on isolated rabbit trachea

Introduction: Dracocephalum kotschyi is a native Iranian plant with antispasmodic activities on smooth muscles such as ileum and uterus. However, so far antispasmodic effect of D. kotschyi on tracheal smooth muscle has not been reported. Therefore, the objective of this research was to investigate antispasmodic activity of D. kotschyi extract and two of its components luteolin and apigenin on rabbit tracheal contraction in vitro. Methods: Rabbits were euthanized by carbon dioxide and the trachea was dissected and immersed in a Tyrode’s solution. Tracheal rings were prepared and mounted vertically in an organ bath at 37°C and gassed continuously with O2. The tracheal ring preparations were contracted with acetylcholine (ACh) and KCl. The isotonic tension was recorded before and after addition of aminophylline, apigenin, luteolin or flavonoids rich extract of D. kotschyi. Flavonoids rich extract were prepared from D. kotschyi using solvent-solvent fractionation technique. Results: Standard drug aminophylline, prevented tracheal ring preparation contracted with ACh. Cumulative addition of aminophylline also attenuated tonic contraction induced by KCl on tracheal smooth muscle. D. kotschyi extract at concentration ranges of 32-512 μg/mL in a concentration dependent manner inhibited KCl and ACh induced tracheal contraction. Apigenin and luteolin (range 16–512 μg/mL) relaxed KCl and ACh-induced contraction of tracheal smooth muscle in vitro in a concentration-dependent manner. Conclusion: This study demonstrated that D. kotschyi extract is a relaxant of tracheal smooth muscle. The relaxant effect of D. kotschyi extract could be due to its flavonoids component such as apigenin and luteolin.