Relaxation Of Tracheal Smooth Muscle Research Articles

Flavones isolated from Pseudognaphalium liebmannii with tracheal smooth muscle relaxant properties

The inflorescences of Pseudognaphalium liebmannii are used as folk medicine to treat various respiratory diseases. In this work, we report the isolation of seven known flavones: 5-hydroxy-3,7-dimethoxyflavone 1, 5,8-dihydroxy-3,7-dimethoxyflavone 2, 5,7-dihydroxy-3,8-dimethoxyflavone 3 (gnaphaliin A), 3,5-dihydroxy-7,8-dimethoxyflavone 4 (gnaphaliin B), 3,5-dihydroxy-6,7,8-trimethoxyflavone 5, 3,5,7-trimethoxyflavone 6 and 3-O-methylquercetin 7. All these flavones except 1 and 6 showed a relaxant effect on guinea pig tracheal preparation with EC50 between 69.91 ± 15.32 and 118.72 ± 7.06 µM. Aminophylline (EC50 = 122.03 ± 7.05 µM) was used as a relaxant reference drug. The active flavones shifted the concentration-response curves of forskolin and nitroprusside leftward, and significantly reduced the EC50 values of these drugs. Furthermore, these flavones dose-dependently inhibited phosphodiesterase (PDE) in an in vitro assay. This reveals that the inflorescences of P. liebmannii contain several flavones with relaxant effect on airway smooth muscle and with PDEs inhibition that contribute to supporting the anti-asthmatic traditional use.

Quercetin supplementation attenuates airway hyperreactivity and restores airway relaxation in rat pups exposed to hyperoxia.

Hyperoxia exposure of immature lungs contributes to lung injury and airway hyperreactivity. Up to now, treatments of airway hyperreactivity induced by hyperoxia exposure have been ineffective. The aim of this study was to investigate the effects of quercetin on hyperoxia-induced airway hyperreactivity, impaired relaxation, and lung inflammation. Newborn rats were exposed to hyperoxia (FiO2 > 95%) or ambient air (AA) for seven days. Subgroups were injected with quercetin (10 mg·kg-1·day-1). After exposures, tracheal cylinders were prepared for in vitro wire myography. Contraction to methacholine was measured in the presence or absence of organ bath quercetin and/or Nω-nitro-L-arginine methyl ester (L-NAME). Relaxation responses were evoked in preconstricted tissues using electrical field stimulation (EFS). Lung tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) levels were measured by enzyme-linked immunosorbent assay (ELISA). A P < 0.05 was considered statistically significant. Contractile responses of tracheal smooth muscle (TSM) of hyperoxic animals were significantly increased compared with AA animals (P < 0.001). Treatment with quercetin significantly reduced contraction in hyperoxic groups compared with hyperoxic control (P < 0.01), but did not have any effect in AA groups. In hyperoxic animals, relaxation of TSM was significantly reduced compared with AA animals (P < 0.001), while supplementation of quercetin restored the lost relaxation in hyperoxic groups. Incubation of preparations in L-NAME significantly reduced the quercetin effects on both contraction and relaxation (P < 0.01). Treatment of hyperoxic animals with quercetin significantly decreased the expression of TNF-α and IL-1β compared with hyperoxic controls (P < 0.001 and P < 0.01, respectively).The findings of this study demonstrate the protective effect of quercetin on airway hyperreactivity and suggest that quercetin might serve as a novel therapy to prevent and treat neonatal hyperoxia-induced airway hyperreactivity and inflammation.

Development of a solid-phase dispersive extraction method for molecularly imprinted polymers and LC-MS/MS for analysis of clenbuterol residues in swine livers and kidneys.

Clenbuterol (CLB) is approved as a veterinary drug because of its tracheal smooth muscle and uterine relaxant effects. However, if improperly administered for the purpose of fattening livestock, CLB can remain in the organs, which may pose a health hazard to humans. We aimed to examine the combination of molecularly imprinted polymer (MIP) and solid-phase dispersive extraction (SPDE) as a pretreatment method for swine liver and kidney, which contain more coexisting impurities than muscle tissue, and attempted to construct an analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Swine livers and kidneys were homogenized and extracted using liquid-liquid partitioning with an ethyl acetate/n-hexane mixture (1:1), followed by SPDE using an MIP gel, and measured using LC-MS/MS. For LC-MS/MS, either an absolute calibration method or isotope dilution mass spectrometry (IDMS) was used. For method validation, a recovery test (additive concentrations: 0.05 and 0.5 ng/g) was conducted, and the data were analyzed using one-way analysis of variance (ANOVA). The recoveries (trueness), repeatability, and intermediate precision obtained using absolute calibration were similar to those obtained using IDMS. Using MIP-SPDE as a pretreatment method for CLB in swine liver and kidney samples yielded comparable results for absolute calibration and IDMS in LC-MS/MS analysis. MIP-SPDE can be used as a pretreatment method to analyze CLB in swine organs with high accuracy.

Impact of the cAMP efflux and extracellular cAMP-adenosine pathway on airway smooth muscle relaxation induced by formoterol and phosphodiesterase inhibitors

β2-adrenoceptors agonists and phosphodiesterase (PDE) inhibitors are effective bronchodilators, due to their ability to increase intracellular cyclic AMP (cAMP) levels and induce airway smooth muscle (ASM) relaxation. We have shown that increment of intracellular cAMP induced by β2-adrenoceptors agonist fenoterol is followed by efflux of cAMP, which is converted by ecto-PDE and ecto-5′-nucleotidases (ecto-5′NT) to adenosine, leading to ASM contraction. Here we evaluate whether other classical bronchodilators used to treat asthma and chronic obstructive pulmonary disease (COPD) could induce cAMP efflux and, as consequence, influence the ASM contractility. Our results showed that β2-adrenoceptor agonists formoterol and PDE inhibitors IBMX, aminophylline and roflumilast induced cAMP efflux and a concentration-dependent relaxation of rat trachea precontracted with carbachol. Pretreatment of tracheas with MK-571 (MRP transporter inhibitor), AMP-CP (ecto-5′NT inhibitor) or CGS-15943 (nonselective adenosine receptor antagonist) potentiated the relaxation induced by β2-adrenoceptor agonists but did not change the relaxation induced by PDE inhibitors. These data showed that all bronchodilators tested were able to induce cAMP efflux. However, only β2-adrenoceptor-induced relaxation of tracheal smooth muscle was affected by cAMP efflux and extracellular cAMP-adenosine pathway.

Rosuvastatin relaxes rat thoracic aorta, pulmonary artery, and trachea via nitric oxide, prostanoids, and potassium channels

Purpose: This study aimed to determine the functional effects and mechanisms of the action of rosuvastatin on vascular and tracheal smooth muscle tissues. &#x0D; Materials and Methods: Vascular and tracheal rings (2-3 mm) isolated from the thoracic aortas, pulmonary arteries, and tracheas of Wistar Albino male rats (250-300 g) were placed in chambers in the isolated tissue bath system. As the resting tension, 1 g was selected. Vascular rings contracted with 10-6 M phenylephrine after a 90-minute equilibration period. Tracheal rings contracted with 10-5 M acetylcholine. After the contraction was steady, rosuvastatin (10-8-10-4 M) was cumulatively applied to the vascular and tracheal rings. The defined experimental methodology was repeated following the incubation of selective inhibitors of signaling pathways and K+ channel blockers to ascertain rosuvastatin's functional effect mechanisms. &#x0D; Results: In the precontracted rat vascular and tracheal rings, rosuvastatin induced concentration-dependent relaxation. The maximal relaxation level in vessel samples was 96%. On the other hand, the maximal relaxation level in tracheal samples was found to be 75%. The vasorelaxant effects of rosuvastatin were dramatically attenuated by endothelium removal, L-NAME treatment, and indomethacin incubation (up to 27%). With the incubation of tetraethylammonium, glyburide, 4-Aminopyridine, and anandamide, rosuvastatin-mediated vascular smooth muscle relaxation levels were significantly decreased (up to 38%). Moreover, With the incubation of tetraethylammonium, glyburide, and 4-Aminopyridine rosuvastatin-mediated tracheal smooth muscle relaxation levels were significantly decreased (up to 30%). &#x0D; Conclusion: Rosuvastatin has a noticeable relaxing effect on the vascular and tracheal smooth muscles. The vasorelaxant effect of rosuvastatin involves intact endothelium, nitric oxide, prostanoids, and K+ channels (BKCa, KV, and KATP channels). Furthermore, nitric oxide, prostanoids, BKCa channels, KV channels, and KATP channels play a role in rosuvastatin-induced tracheal smooth muscle relaxation

Pituitary Adenylate Cyclase Activating Peptide Restores Hyperoxia-Impaired Relaxation of Tracheal Smooth Muscle in Neonatal Rats

Neonatal hyperoxia induces airway hyperreactivity by potentiating contractile responses and impairing the relaxation of airway smooth muscle (ASM). Maintenance of balance between these processes in airways is critical for normal flow of air, and non-adrenergic- noncholinergic inhibitory system is an important component of airway smooth muscle tone. Therefore, we tested the hypothesis that exogenous pituitary adenylate cyclase activating peptide (PACAP) will restore the impaired relaxant responses of tracheal smooth muscle exposed to neonatal hyperoxia. Tracheal cylinders were obtained from Wistar rat pups (P10) exposed to hyperoxia (≥ 95% O2) or room air for seven days. These cylinders were used to study relaxant responses of ASM evoked by electrical field relaxation (EFS) in precontracted tissues (carbachol, 5x10μM) in absence or presence of a single dose of PACAP (50 nM). In addition, the dose-response relaxant effect of PACAP (1nM – 1μM) was tested in absence or presence of a PAC1 receptor antagonist; a protein kinase A (PKA) inhibitor (Rp-8-CPT-cAMPS); a phosphodiesterase 4 (PDE4) inhibitor (rolipram); or a large-conductance calcium-activated potassium channel blocker (charybdotoxin). The relaxation is expressed as percentage of preconstricted state, and data are presented as mean±s.e.m. Hyperoxic exposure significantly decreased the relaxant responses of ASM towards EFS, as compared to those obtained from control animals exposed to room air, and these reduced relaxant responses in hyperoxic tissues were significantly reversed in presence of PACAP (p&lt;0.001). PACAP induced dose-dependent relaxation and the relaxant responses were overcompensated in hyperoxic animals. PAC1 antagonist; PKA; PDE4 inhibition; or blockade of calcium-activated potassium channel significantly reduced PACAP-induced relaxation (p&lt;0.01).The results of this study revealed that PACAP reverses the impaired relaxation of airway smooth muscle induced by hyperoxic exposure via cAMP/PKA signaling pathway and through calcium-activated potassium channel. We speculate that use of PACAP might be an effective therapeutic approach to prevent the airway hyperreactivity induced by hyperoxia. Supported by the Ministry of Education, Science, and Technology of the Republic of Kosovo - Small Research Grants: 2-2459-11 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Elabela relaxes rat pulmonary artery and trachea via BKCa, KV, and KATP channels

ObjectiveElabela is a newly discovered peptide hormone. This study aimed to determine the functional effects and mechanisms of action of elabela in rat pulmonary artery and trachea. Materials and methodsVascular rings isolated from the pulmonary arteries of male Wistar Albino rats were placed in chambers in the isolated tissue bath system. The resting tension was set to 1 g. After the equilibration period, the pulmonary artery rings were contracted with 10-6 M phenylephrine. Once a stable contraction was achieved, elabela was applied cumulatively (10-10-10-6 M) to the vascular rings. To determine the vasoactive effect mechanisms of elabela, the specified experimental protocol was repeated after the incubation of signaling pathway inhibitors and potassium channel blockers. The effect and mechanisms of action of elabela on tracheal smooth muscle were also determined by a similar protocol. ResultsElabela exhibited a concentration-dependent relaxation in the precontracted rat pulmonary artery rings (p < .001). Maximal relaxation level was 83% (pEC50: 7.947 CI95(7.824–8.069)). Removal of the endothelium, indomethacin incubation, and dideoxyadenosine incubation significantly decreased the vasorelaxant effect levels of elabela (p < .001). Elabela-induced vasorelaxation levels were significantly reduced after iberiotoxin, glyburide, and 4-Aminopyridine administrations (p < .001). L-NAME, methylene blue, apamin, TRAM-34, anandamide, and BaCl2 administrations did not cause a significant change in the vasorelaxant effect level of elabela (p = 1.000). Elabela showed a relaxing effect on precontracted tracheal rings (p < .001). Maximal relaxation level was 73% (pEC50: 6.978 CI95(6.791–7.153)). The relaxant effect of elabela on tracheal smooth muscle was decreased significantly after indomethacin, dideoxyadenosine, iberiotoxin, glyburide, and 4-Aminopyridine incubations (p < .001). ConclusionsElabela exerted a prominent relaxant effect in the rat pulmonary artery and trachea. Intact endothelium, prostaglandins, cAMP signaling pathway, and potassium channels (BKCa, KV, and KATP channels) are involved in the vasorelaxant effect of elabela. Prostaglandins, cAMP signaling pathway, BKCa channels, KV channels, and KATP channels also contribute to elabela-induced tracheal smooth muscle relaxant effect.

Design of Multitarget Inhibitors as Tracheal Smooth Muscle Relaxants.

Asthma complications and adverse effects associated with steroidal therapy highlight the need for non-steroidal compounds intercepting asthmatic pathophysiology at multiple targets. The present investigation was carried out to evaluate the tracheal smooth muscle relaxant effect of virtually designed, combinatorially synthesized polyfunctional N-heteroarylamides. Virtual screening and molecular docking studies of designed compounds were performed using PyRx and AUTODOCK 4.2 software against molecular targets viz. FLAP, LTB4, and H1 receptor. Cross-validation of virtual screening results and active site, confirmation was performedusingVlife MDS software version 3.5. The combinatorial approach was used to synthesize designed compounds in which heterocyclic amines were reacted with substituted aromatic acid chlorides by nucleophilic substitution reaction to obtain a 5x5 mini-library. The structures of synthesized leads were confirmed by infrared and proton magnetic resonance spectroscopic analysis. Synthesized compounds were evaluated for their smooth muscle relaxation effect on isolated goat tracheal smooth muscle. Results were calculated as a percent decrease in contraction response observed using histamine and LTB4. The tested compounds produced anticipated tracheal smooth muscle relaxant activity. Based on the results of screening the structure-activity relationships (SAR) have been reported. Present study concluded that synthesized polyfunctional N-heteroarylamides have a tracheal smooth muscle relaxant effect. The mode of action is predicted from the analysis of virtual screening results. A good correlation was observed between virtual screenings and biological activities of lead molecules suggesting the rationale used to optimize the structural requirements of a ligand for selected targets is appropriate.

Effects of Allium cepa and Its Constituents on Respiratory and Allergic Disorders: A Comprehensive Review of Experimental and Clinical Evidence.

The health benefits of Allium cepa (A. cepa) have been proclaimed for centuries. Various pharmacological and therapeutic effects on respiratory, allergic, and immunologic disorders are shown by A. cepa and its constituents. Flavonoids such as quercetin and kaempferol, alk(en)yl cysteine sulfoxides including S-methyl cysteine sulfoxide and S-propyl cysteine sulfoxide, cycloalliin, thiosulfinates, and sulfides are the main compounds of the plant. A. cepa displays broad-spectrum pharmacological activities including antioxidant, anti-inflammatory, antihypertensive, and antidiabetic effects. Our objective in this review is to present the effects of A. cepa and its constituents on respiratory, allergic, and immunologic disorders. Different online databases were searched to find articles related to the effect of A. cepa extracts and its constituents on respiratory, allergic, and immunologic disorders until the end of December 2020 using keywords such as onion, A. cepa, constituents of A. cepa, therapeutic effects and pharmacological effects, and respiratory, allergic, and immunologic disorders. Extracts and constituents of A. cepa showed tracheal smooth muscle relaxant effects, indicating possible bronchodilator activities or relieving effects on obstructive respiratory diseases. In experimental animal models of different respiratory diseases, the preventive effect of various extracts and constituents of A. cepa was induced by their antioxidant, immunomodulatory, and anti-inflammatory effects. The preventive effects of the plant and its components on lung disorders induced by exposure to noxious agents as well as lung cancer, lung infection, and allergic and immunologic disorders were also indicated in the experimental and clinical studies. Therefore, this review may be considered a scientific basis for development of therapies using this plant, to improve respiratory, allergic, and immunologic disorders.

Evaluation of the Antinociceptive, Antiallodynic, Antihyperalgesic and Anti-Inflammatory Effect of Polyalthic Acid.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are very commonly used, but their adverse effects warrant investigating new therapeutic alternatives. Polyalthic acid, a labdane-type diterpenoid, is known to produce gastroprotection, tracheal smooth muscle relaxation, and antitumoral, antiparasitic and antibacterial activity. This study aimed to evaluate the antinociceptive, antiallodynic, antihyperalgesic and anti-inflammatory effect of polyalthic acid on rats. Moreover, the effectiveness of treating hyperalgesia with a combination of polyalthic acid and naproxen was analyzed, as well as the type of drug–drug interaction involved. Nociception was examined by injecting 1% formalin into the right hind paw and thermal hyperalgesia and inflammation by injecting a 1% carrageenan solution into the left hind paw of rats. Allodynia was assessed on an L5/L6 spinal nerve ligation model. Polyalthic acid generated significant antinociceptive (56–320 mg/kg), antiallodynic (100–562 mg/kg), and antihyperalgesic and anti-inflammatory (10–178 mg/kg) effects. Antinociception mechanisms were explored by pretreating the rats with naltrexone, ODQ and methiothepin, finding the effect blocked by the former two compounds, which indicates the participation of opioid receptors and guanylate cyclase. An isobolographic analysis suggests synergism between polyalthic acid and naproxen in the combined treatment of hyperalgesia.

Regulation of smooth muscle contractility by the epithelium in rat tracheas: role of prostaglandin E2 induced by the neurotransmitter acetylcholine.

BackgroundPrevious studies have suggested the involvement of epithelium in modulating the contractility of neighboring smooth muscle cells. However, the mechanism underlying epithelium-derived relaxation in airways remains largely unclear. This study aimed to investigate the mechanism underlying epithelium-dependent smooth muscle relaxation mediated by neurotransmitters.MethodsThe contractile tension of Sprague-Dawley (SD) rat tracheal rings were measured using a mechanical recording system. Intracellular Ca2+ level was measured using a Ca2+ fluorescent probe Fluo-3 AM, and the fluorescence signal was recorded by a laser scanning confocal imaging system. The prostaglandin E2 (PGE2) content was measured using an enzyme-linked immunosorbent assay kit.ResultsWe observed that the neurotransmitter acetylcholine (ACh) restrained the electric field stimulation (EFS)-induced contraction in the intact but not epithelium-denuded rat tracheal rings. After inhibiting the muscarinic ACh receptor (mAChR) or cyclooxygenase (COX), a critical enzyme in prostaglandin synthesis, the relaxant effect of ACh was attenuated. Exogenous PGE2 showed a similar inhibitory effect on the EFS-evoked contraction of tracheal rings. Moreover, ACh triggered phospholipase C (PLC)-coupled Ca2+ release from intracellular Ca2+ stores and stimulated COX-dependent PGE2 production in primary cultured rat tracheal epithelial cells.ConclusionsCollectively, this study demonstrated that ACh induced rat tracheal smooth muscle relaxation by promoting PGE2 release from tracheal epithelium, which might provide valuable insights into the cross-talk among neurons, epithelial cells and neighboring smooth muscle cells in airways.

The Relaxant Effect of Plantago Major on Rat Tracheal Smooth Muscles and Its Possible Mechanisms.

This study was conducted to evaluate the possible mechanisms of the relaxant effects of hydroalcoholic extract of Plantago major (P. major) on tracheal smooth muscle (TSM) in rats. The effects of cumulative concentrations of P. major (5, 10, 20 and 40 mg/mL) and theophylline (0.2, 0.4, 0.6 and 0.8 mM) were evaluated on pre-contracted TSM with 10 μΜ methacholine or 60 mM KCl. To determine the possible mechanisms, the relaxant effect of the plant was also examined on incubated TSM with atropine, indomethacin, chlorpheniramine, glibenclamide, diltiazem, papaverine, and propranolol. The results indicated concentration-dependent relaxant effects for P. major in non-incubated TSM contracted by methacholine or KCl. There was no statistically significant difference in the relaxant effects of P. major between non-incubated and incubated tissues with indomethacin, papaverine, and propranolol. However, the relaxant effects of P. major in incubated tissues with atropine (p<0.01 to p<0.001), chlorpheniramine (p<0.05 to p<0.001), glibenclamide (p<0.05), or diltiazem (p<0.01) were significantly lower than non-incubated TSM. P. major indicated relatively potent relaxant effects which were lower than those of theophylline. Muscarinic and histamine (H1) receptors inhibition, as well as calcium channel blocking and potassium channel opening effects are suggested to contribute to the TSM relaxant effect of the plant.

Curcumin analogs (B2BrBC and C66) supplementation attenuates airway hyperreactivity and promote airway relaxation in neonatal rats exposed to hyperoxia.

BackgroundThis study was undertaken to test the hypothesis that the newly synthesized curcuminoids B2BrBC and C66 supplementation will overcome hyperoxia‐induced tracheal hyperreactivity and impairment of relaxation of tracheal smooth muscle (TSM).Materials and methodsRat pups (P5) were exposed to hyperoxia (>95% O2) or normoxia for 7 days. At P12, tracheal cylinders were used to study in vitro contractile responses induced by methacholine (10−8–10−4M) or relaxation induced by electrical field stimulation (5–60 V) in the presence/absence of B2BrBC or C66, or to study the direct relaxant effects elicited by both analogs.ResultsHyperoxia significantly increased contraction and decreased relaxation of TSM compared to normoxia controls. Presence of B2BrBC or C66 normalized both contractile and relaxant responses altered by hyperoxia. Both, curcuminoids directly induced dose‐dependent relaxation of preconstricted TSM. Supplementation of hyperoxic animals with B2BrBC or C66, significantly increased catalase activity. Lung TNF‐α was significantly increased in hyperoxia‐exposed animals. Both curcumin analogs attenuated increases in TNF‐α in hyperoxic animals.ConclusionWe show that B2BrBC and C66 provide protection against adverse contractility and relaxant effect of hyperoxia on TSM, and whole lung inflammation. Both analogs induced direct relaxation of TSM. Through restoration of catalase activity in hyperoxia, we speculate that analogs are protective against hyperoxia‐induced tracheal hyperreactivity by augmenting H2O2 catabolism. Neonatal hyperoxia induces increased tracheal contractility, attenuates tracheal relaxation, diminishes lung antioxidant capacity, and increases lung inflammation, while monocarbonyl CUR analogs were protective of these adverse effects of hyperoxia. Analogs may be promising new therapies for neonatal hyperoxic airway and lung disease.

Calcium and potassium channels are involved in curcumin relaxant effect on tracheal smooth muscles

Context Curcumin, the active component of Curcuma longa L. (Zingiberaceae), exhibits a wide variety of biological activities including vasodilation and anti-inflammation. Objective The relaxant effect of curcumin in tracheal smooth muscle (TSM) was not examined so far, thus, this study was designed to assess the relaxant effect of curcumin on rat TSM and examine the underlying mechanism(s) responsible for this effect. Materials and methods TSM was contracted by KCl (60 mM) or methacholine (10 μM), and cumulative concentrations of curcumin (12.5, 25, 50, and 100 mg/mL) or theophylline (0.2, 0.4, 0.6, and 0.8 mM, as positive control) were added to organ bath. The relaxant effect of curcumin was examined in non-incubated or incubated tissues with atropine (1 μM), chlorpheniramine (1 μM), indomethacin (1 μM), and papaverine (100 μM). Results In non-incubated TSM, curcumin showed significant relaxant effects on KCl-induced contraction in a concentration-dependent manner (p < 0.001 for all concentrations). The relaxant effects of curcumin 12.5, 25, and 50 mg/mL were significantly lower in atropine-incubated tissue compared to non-incubated TSM (p < 0.05 to p < 0.001). A significant difference was observed in EC50 between atropine-incubated (48.10 ± 2.55) and non-incubated (41.65 ± 1.81) tissues (p < 0.05). Theophylline showed a significant relaxant effect on both KCl and methacholine-induced contraction in a concentration-dependent manner (p < 0.001 for all cases). Conclusions The results indicated a relatively potent relaxant effect of curcumin on TSM, which was less marked than the effect of theophylline. Calcium channel blocking and/or potassium channel opening properties of curcumin may be responsible for TSM relaxation.

Histamine (H1) Receptors, Cyclooxygenase Pathway and Nitric Oxide Formation Involved in Rat Tracheal Smooth Muscle Relaxant Effect of Berberine.

In this study we aimed to examine the relaxant effect of berberine, a compound extracted from a variety of herbs, on rat tracheal smooth muscle (TSM) and its possible mechanism(s). Cumulative concentrations of berberine (20, 65, 200 and 600 μg/mL) were added on pre-contracted TSM by methacholine or KCl in non-incubated or incubated tissues with atropine, chlorpheniramine, propranolol, diltiazem, glibenclamide, indomethacin, L-NG-nitro arginine methyl ester (L-NAME) and papaverine. The relaxant effects of theophylline (0.2, 0.4, 0.6 and 0.8 mM) as positive control and saline (1 mL) as negative control were also examined in non-incubated tissues. Berberine showed significant and concentration-dependent relaxant effects in non-incubated tissues contracted by KCl and methacholine (p<0.01 to p<0.001). There was no significant difference in the relaxant effects of berberine between non-incubated and incubated tissues with atropine, propranolol, diltiazem, glibenclamide, and papaverine. The relaxant effects of second concentrations of berberine in incubated tissues with L-NAME, its three lower concentration in incubated tissues with chlorpheniramine and its all concentrations in incubated tissues with indomethacin were significantly lower than non-incubated tissues (p<0.05 to p<0.001). The EC50 values of berberine in incubated tissues with chlorpheniramine was significantly higher than the non-incubated condition (p<0.05). Our findings reveal a relatively potent relaxant effect of berberine that is lower than the effect of theophylline. Proposed mechanisms for the relaxant effect of berberine are histamine (H1) receptor blockade, inhibition of cyclooxygenase pathways and/or nitric oxide formation.

Diphenethylpiperidine alkaloids with tracheal smooth muscle relaxation activity from Hippobroma longiflora (L.) G. Don

Phytochemical investigations on the whole plant of Hippobroma longiflora (L.) G. Don have resulted in the isolation and elucidation of three new diphenethylpiperidine alkaloids, hippofoline A (1), hippofoline B (2), and (−)-cis-2′,2′′-diphenyllobelidiol N-oxide (5), alongside two known alkaloids, (−)-lobeline (3) and (−)-cis-2′,2′′-diphenyllobelidiol (4). The absolute configurations of alkaloids 1 and 2 were deduced based on experimental and calculated ECD data as well as biogenetic consideration, while the absolute configurations of alkaloids 3 and 4 in their free base form were confirmed by Cu Kα radiation X-ray diffraction analyses. Alkaloids 2–4 were shown to induce concentration-dependent relaxation effects on rat isolated tracheal rings that were pre-contracted with carbachol.

Discovery of β-arrestin-biased β2-adrenoceptor agonists from 2-amino-2-phenylethanol derivatives.

β-Arrestins are a small family of proteins important for signal transduction at G protein-coupled receptors (GPCRs). β-Arrestins are involved in the desensitization of GPCRs. Recently, biased ligands possessing different efficacies in activating the G protein- versus the β-arrestin-dependent signals downstream of a single GPCR have emerged, which can be used to selectively modulate GPCR signal transduction in such a way that desirable signals are enhanced to produce therapeutic effects while undesirable signals of the same GPCR are suppressed to avoid side effects. In the present study, we evaluated agonist bias for compounds developed along a drug discovery project of β2-adrenoceptor agonists. About 150 compounds, including derivatives of fenoterol, 2-amino-1-phenylethanol and 2-amino-2-phenylethanol, were obtained or synthesized, and initially screened for their β-adrenoceptor-mediated activities in the guinea pig tracheal smooth muscle relaxation assay or thecardiomyocyte contractility assay. Nineteen bioactive compounds were further assessed using both the HTRF cAMP assay and the PathHunter β-arrestin assay. Their concentration-response data in stimulating cAMP synthesis and β-arrestin recruitment were applied to the Black-Leff operational model for ligand bias quantitation. As a result, three compounds (L-2, L-4, and L-12) with the core structure of 5-(1-amino-2-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one were identified as a new series of β-arrestin-biased β2-adrenoceptor agonists, whereas salmeterol was found to be Gs-biased. These findings would facilitate the development of novel drugs for the treatment of both heart failure and asthma.

PL-015 Aerobic exercise increases BKCa channel expression to enhance tracheal smooth muscle relaxation in a murine asthma model

Objective Increasing evidence has shown that moderate-intensity aerobic exercise training reduces airway hyperresponsiveness (AHR) in patients with asthma. However, the mechanisms underlying exercise-induced improvements in smooth muscle contractility have not been fully elucidated. Large-conductance Ca2+-activated K+ channels (BKCa) are expressed broadly on smooth muscle cells and play an important role in the regulation of smooth muscle contraction. We tested the hypothesis that exercise training increases the contribution of BKCa channel to tracheal smooth muscle relaxation in in ovalbumin (OVA)-challenged asthmatic rats.&#x0D; Methods Rats were sensitized/challenged with OVA or saline and exercised at a moderate intensity 5 times/week for 4 weeks. Tracheal smooth muscle contractility was tested. Membrane potential of primary cultured tracheal smooth muscle cells was measured. In addition, western immunoblotting was performed to study the expression levels of BKCa channel protein.&#x0D; Results The contraction of rat airway smooth muscle induced by carbachol was significantly increased with asthma and exercise training reversed this alteration. Application of BKCa channel agonist, NS1619, induced tracheal smooth muscle relaxation. NS1619-induced relaxation was decreased in asthmatic rats, however exercise training significantly increased NS1619-induced relaxation. In primary cultured smooth muscle cells, NS1619-induced membrane potential was reduced with asthma and this alteration was diminished after exercise training. Additionally, western blotting revealed that the protein expression of BKCa was reduced in asthmatic group and aerobic exercise significantly improved BKCa expression.&#x0D; Conclusions The present study reveals that aerobic exercise training increases BKCa expression on tracheal smooth muscle, which partly underlies the beneficial effect of exercise on improving airway smooth muscle relaxation in asthma.

Muscarinic receptors, nitric oxide formation and cyclooxygenase pathway involved in tracheal smooth muscle relaxant effect of hydro-ethanolic extract of Lavandula angustifolia flowers

BackgroundLavandula angustifolia (L. angustifolia) Mill. (Common name Lavender) is used in traditional and folk medicines for the treatment of various diseases including respiratory disorders worldwide. The relaxant effect of the plant on the smooth muscle of some tissues was shown previously. The present study has investigated the role of different receptors and pathways in the relaxant effect of L. angustifolia on tracheal smooth muscle. MethodsCumulative concentrations of the hydro-ethanolic extract of L. angustifolia flowers (0.5, 1, 2 and 4 mg/ml) were added on pre-contracted tracheal smooth muscle by methacholine (10 μM) or KCl (60 mM) on non-preincubated or preincubated tissues with atropine, chlorpheniramine, propranolol, diltiazem, glibenclamide, indomethacin, ω-nitro-L-arginine methyl ester (L-NAME) and papaverine. The results compared with of theophylline (0.2, 0.4, 0.6 and 0.8 mM) as positive control and saline (1 ml) as negative control. ResultsThe extract showed concentration-dependent relaxant effects in non-preincubated tracheal smooth muscle contracted by KCl and methacholine (p < 0.05 to p < 0.001). The relaxant effect ofL. angustifolia was not significantly different between non-preincubated and preincubated tissues with chlorpheniramine, propranolol, diltiazem, glibenclamide, and papaverine. However, two higher concentrations of L. angustifolia in preincubated tissues with L-NAME (p < 0.01), indomethacin (p < 0.05 to p < 0.001) and atropine (p < 0.05) showed significantly lower relaxant effects than non-preincubated tissues. The EC50 values of L. angustifolia in tissues preincubated with indomethacin was significantly higher than non-preincubated trachea (p < 0.05). The effects of three first concentrations of the extract on KCl and methacholine-induced muscle contraction were significantly lower than those of theophylline (p < 0.05 to p < 0.001). ConclusionsThese results indicated a relatively potent relaxant effect ofL. angustifolia that was lower than the effect of theophylline. The possible mechanisms of relaxant effect of this plant on tracheal smooth muscle are muscarinic receptors blockade, inhibition of cyclooxygenase pathways and/or involvement of nitric oxide production. Its clinical applications should be investigated in further studies.

Relaxant effect of ghrelin on guinea pig isolated tracheal smooth muscle: role of epithelial NO and PGE2.

This study aimed at investigating the potential ghrelin relaxing effect on guinea pig isolated tracheal smooth muscle (TSM). Using an in vitro experimental approach, the physiological role of the airway epithelium on smooth muscle relaxation has been investigated by analyzing the dose-response curves for carbachol- or histamine-induced contractions on epithelium intact versus denuded tracheal tissue. The relaxant effect of ghrelin (5-200μmol/L) then investigated on carbachol-contracted, non-sensitized, and ovalbumin (OVA)-sensitized guinea pig TSM with an intact or denuded epithelium. The isolated TSMs from identical guinea pigs were incubated in Krebs solution aerated with 95% O2 and 5% CO2 through an automated tissue organ bath system (n = 6 for each group). The ghrelin relaxation mechanism was assessed by adding L-NAME, indomethacin, and YIL-781 for GHS-R1 into the tissue chamber. The spasmogens carbachol and histamine have shown a significantly higher contracting effect on epithelium-denuded than in epithelium-intact TSM confirmed by the significantly higher mean pEC50 of both agonists on the epithelium-denuded trachea (p < 0.05). Ghrelin has shown a concentration-dependent relaxing effect on carbachol-contracted TSM (r = 0.96, p = 0.00). The effect was more evident in the intact non-sensitized than in epithelium-denuded or OVA-sensitized groups (p < 0.05). Preincubation with nitric oxide (NO) and prostaglandin E2 (PGE2) inhibitors has significantly reduced the ghrelin-induced relaxation on epithelium-intact TSM suggesting an epithelium-dependant mechanism. However, GHS-R1a antagonist has also succeeded to reduce ghrelin relaxant effect, which needs further clarification. Ghrelin proved to have a potential TSM relaxant effect possibly through epithelium-dependant mechanisms involving NO and PGE2.