Guinea Pig Tracheal Smooth Muscle Research Articles

17β-estradiol induces hyperresponsiveness in guinea pig airway smooth muscle by inhibiting the plasma membrane Ca2+-ATPase

High serum estrogen concentrations are associated with asthma development and severity, suggesting a link between estradiol and airway hyperresponsiveness (AHR). 17β-estradiol (E2) has non-genomic effects via Ca2+ regulatory mechanisms; however, its effect on the plasma membrane Ca2+-ATPases (PMCA1 and 4) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) is unknown. Hence, in the present study, we aim to demonstrate if E2 favors AHR by increasing intracellular Ca2+ concentrations in guinea pig airway smooth muscle (ASM) through a mechanism involving Ca2+-ATPases.In guinea pig ASM, Ca2+ microfluorometry, muscle contraction, and Western blot were evaluated. Then, we performed molecular docking analysis between the estrogens and Ca2+ ATPases.In tracheal rings, E2 produced AHR to carbachol. In guinea pig myocytes, acute exposure to physiological levels of E2 modified the transient Ca2+ peak induced by caffeine to a Ca2+ plateau. The incubation with PMCA inhibitors (lanthanum and carboxyeosin, CE) partially reversed the E2-induced sustained plateau in the caffeine response. In contrast, cyclopiazonic acid (SERCA inhibitor), U-0126 (an inhibitor of ERK 1/2), and choline chloride did not modify the Ca2+ plateau produced by E2. The mitochondrial uniporter activity and the capacitative Ca2+ entry were unaffected by E2. In guinea pig ASM, Western blot analysis demonstrated PMCA1 and PMCA4 expression. The results from the docking modeling demonstrate that E2 binds to both plasma membrane ATPases. In guinea pig tracheal smooth muscle, inhibiting the PMCA with CE, induced hyperresponsiveness to carbachol. 17β-estradiol produces hyperresponsiveness by inhibiting the PMCA in the ASM and could be one of the mechanisms responsible for the increase in asthmatic crisis in women.

Theophylline-Induced Relaxation Is Enhanced after Testosterone Treatment via Increased KV1.2 and KV1.5 Protein Expression in Guinea Pig Tracheal Smooth Muscle.

Theophylline is a drug commonly used to treat asthma due to its anti-inflammatory and bronchodilatory properties. Testosterone (TES) has been suggested to reduce the severity of asthma symptoms. This condition affects boys more than girls in childhood, and this ratio reverses at puberty. We reported that guinea pig tracheal tissue chronic exposure to TES increases the expression of β2-adrenoreceptors and enhances salbutamol-induced K+ currents (IK+). Herein, we investigated whether the upregulation of K+ channels can enhance the relaxation response to methylxanthines, including theophylline. Chronic incubation of guinea pig tracheas with TES (40 nM, 48 h) enhanced the relaxation induced by caffeine, isobutylmethylxanthine, and theophylline, an effect that was abolished by tetraethylammonium. In tracheal myocytes, chronic incubation with TES increased theophylline-induced IK+; flutamide reversed this effect. The increase in IK+ was blocked by 4-aminopyridine by ~82%, whereas iberiotoxin reduced IK+ by ~17%. Immunofluorescence studies showed that chronic TES exposure increased the expression of KV1.2 and KV1.5 in airway smooth muscle (ASM). In conclusion, chronic exposure to TES in guinea pig ASM promotes upregulation of KV1.2 and KV1.5 and enhances theophylline relaxation response. Therefore, gender should be considered when prescribing methylxanthines, as teenage boys and males are likely to respond better than females.

Effects of NP-1815-PX, a P2X4 Receptor Antagonist, on Contractions in Guinea Pig Tracheal and Bronchial Smooth Muscles.

Administration of a P2X4 receptor antagonist to asthma model mice improved asthma symptoms, suggesting that P2X4 receptor antagonists may be new therapeutics for asthma. However, the effects of these antagonists on tracheal/bronchial smooth muscle (TSM and BSM) have not been investigated. This study examined the effects of NP-1815-PX, a selective P2X4 receptor antagonist, on guinea pig TSM and BSM contractions. In epithelium-intact TSM, NP-1815-PX (10-5 M) strongly suppressed ATP-induced contractions. ATP-induced contractions were strongly suppressed by indomethacin (3 × 10-6 M) and ONO-8130 (a prostanoid EP1 receptor antagonist, 10-7 M). ATP-induced contractions were partially suppressed by SQ 29,548 (a prostanoid TP receptor antagonist, 3 × 10-7 M), although the difference was not significant. In contrast, ATP-induced contractions were not affected by AL 8810 (a prostanoid FP receptor antagonist, 10-5 M) or L-798,106 (a prostanoid EP3 receptor antagonist, 10-8 M). NP-1815-PX (10-5-10-4 M) strongly suppressed U46619 (a TP receptor agonist)- and prostaglandin F2α (PGF2α)-induced epithelium-denuded TSM and BSM contractions, which were largely inhibited by SQ 29,548. Additionally, NP-1815-PX (10-5-10-4 M) strongly suppressed the U46619-induced increase in intracellular Ca2+ concentrations in human TP receptor-expressing cells. However, NP-1815-PX (10-4 M) did not substantially inhibit the TSM/BSM contractions induced by carbachol, histamine, neurokinin A, or 50 mM KCl. These findings indicate that NP-1815-PX inhibits guinea pig TSM and BSM contractions mediated through the TP receptor, in addition to the P2X4 receptor, whose stimulation mainly induces EP1 receptor-related mechanisms. Thus, these findings support the usefulness of NP-1815-PX as a therapeutic drug for asthma.

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.

Docosahexaenoic acid (DHA) selectively inhibits prostanoid TP receptor-mediated contractions of guinea pig tracheal smooth muscle

Docosahexaenoic acid (DHA) selectively inhibits prostanoid TP receptor-mediated contractions of guinea pig tracheal smooth muscle

Effect of NP-1815-PX (a P2X4 receptor antagonist) on the contraction of guinea pig tracheal smooth muscles

Effect of NP-1815-PX (a P2X4 receptor antagonist) on the contraction of guinea pig tracheal smooth muscles

Leptin induces a contracting effect on guinea pig tracheal smooth muscle via the Ob-R receptor mechanism: novel evidence.

The purpose of this study was to explore the potential contracting effect of leptin on isolated guinea pig tracheal smooth muscle (TSM), the possible mechanism, and the impact of epithelium denudation or allergen sensitization, respectively. An in vitro experiment investigated the effect of leptin at a concentration of 250-1000 nmol/L on isolated guinea pig TSM with an intact or denuded epithelium. Ovalbumin and IgE were used to test the impact of active and passive sensitization. The isolated TSM strips were incubated in Krebs solution and aerated with carbogen (95% O2 and 5% CO2) via an automated tissue organ bath system (n = 4 for each group). Isometric contractions were recorded digitally using iox2 data acquisition software. The possible mechanism of leptin-induced TSM contraction was examined by preincubation with leptin receptor (Ob-R) antagonist. Leptin had significant concentration-dependent contraction effects on guinea pig TSM (p < 0.05). Epithelium denuding and active or passive sensitization significantly increased the potency of the leptin. Preincubation with a leptin receptor (Ob-R) antagonist significantly reduced the contraction effects, suggesting an Ob-R-mediated mechanism. Leptin had a contracting effect on airway smooth muscles potentiated by either epithelium denuding or sensitization, and the Ob-R mechanism was a possible effect mediator.

Synthesis, Purification, and Selective β2-AR Agonist and Bronchodilatory Effects of Catecholic Tetrahydroisoquinolines from Portulaca oleracea

A green, biomimetic, phosphate-mediated Pictet-Spengler reaction was used in the synthesis of three catecholic tetrahydroisoquinolines, 1, 2, and 12, present in the medicinal plant Portulaca oleracea, as well as their analogues 3-11, 13, and 14, with dopamine hydrochloride and aldehydes as the substrates. AB-8 macroporous resin column chromatography was applied for purification of the products from the one-step high-efficacy synthesis. It eliminated the difficulties in the isolation of catecholic tetrahydroisoquinolines from the aqueous reaction system and unreacted dopamine hydrochloride. Activity screening in CHO-K1/Gα15 cell models consistently expressing α1B-, β1-, or β2-adrenergic receptors indicated that 12 and 2, compounds that are present in P. oleracea, possessed the most potent β2-adrenergic receptor agonist activity and 2 was a selective β2-adrenergic receptor agonist at the concentration of 100 μM. Both 12 and 2 exhibited dose-dependent bronchodilator effects on the histamine-induced contraction of isolated guinea-pig tracheal smooth muscle, with EC50 values of 0.8 and 2.8 μM, respectively. These findings explain the scientific rationale of P. oleracea use as an antiasthmatic herb in folk medicine and provide the basis for the discovery of novel antiasthma drugs.

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.

A novel GABAA receptor ligand MIDD0301 with limited blood-brain barrier penetration relaxes airway smooth muscle ex vivo and in vivo.

Airway smooth muscle (ASM) cells express GABA A receptors (GABAARs), and previous reports have demonstrated that GABAAR activators relax ASM. However, given the activity of GABAARs in central nervous system inhibitory neurotransmission, concern exists that these activators may lead to undesirable sedation. MIDD0301 is a novel imidazobenzodiazepine and positive allosteric modulator of the GABAAR with limited brain distribution, thus eliminating the potential for sedation. Here, we demonstrate that MIDD0301 relaxes histamine-contracted guinea pig ( P < 0.05, n = 6-9) and human ( P < 0.05, n = 6-10) tracheal smooth muscle ex vivo in organ bath experiments, dilates mouse peripheral airways ex vivo in precision-cut lung-slice experiments ( P < 0.001, n = 16 airways from three mice), and alleviates bronchoconstriction in vivo in mice, as assessed by the forced-oscillation technique ( P < 0.05, n = 6 mice). Only trace concentrations of the compound were detected in the brains of mice after inhalation of nebulized 5 mM MIDD0301. Given its favorable pharmacokinetic properties and demonstrated ability to relax ASM in a number of clinically relevant experimental paradigms, MIDD0301 is a promising drug candidate for bronchoconstrictive diseases, such as asthma.

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.

Testosterone induces hyporesponsiveness by interfering with IP3 receptors in guinea pig airway smooth muscle.

Asthma symptoms have been associated with sex steroids. During childhood, this illness seems more frequent in boys than in girls and this tendency reverts in puberty when it is more severe in women. Testosterone (TES), at supraphysiological concentrations, relaxed pre-contracted airway smooth muscle, but its effects at physiological concentrations have not been thoroughly studied. We explored this possibility in guinea pig tracheal smooth muscle. In myocytes TES (10 nM) abolished carbachol (CCh)-induced intracellular Ca2+ concentration ([Ca2+]i) increment. Ca2+ responses to ATP were partially modified by TES while histamine's were not. These results indicate that inositol 1,4,5-trisphosphate (IP3) signaling pathway might be involved. Photolysis of caged-IP3 increased [Ca2+]i and TES abolished this effect. TES diminished reactivity of the smooth muscle to CCh and this effect was non-genomic since it was unchanged by flutamide. In tracheal smooth muscle, mRNA for each IP3 receptor (ITPR) isoform was found and, by immunofluorescence, ITPR1 and ITPR3 seems to be the main isoforms observed while ITPR2 was less prominent. Comparing the amino acid sequence of ITPR1 and the sequence of the TES binding site on the androgen receptor, we found that they share a short sequence. This domain could be responsible for the TES binding to the ITPR1 and probably for its blocking effect. We conclude that TES modifies ITPR1 function in airway smooth muscle, turning this tissue less reactive to contractile agonists that act through PLCβ-IP3 signaling cascade. These results might be related to the low asthma prevalence in males from puberty to adulthood.

Hydro-ethanolic Extract of Portulaca oleracea Affects Beta-adrenoceptors of Guinea Pig Tracheal Smooth Muscle.

Thestimulatory effect of the extract of Portulaca oleracea (P. olerace) on β-adrenoceptor of tracheal smooth muscle was examined.To examine β-adrenoceptor stimulatory effect, concentration response curve to isoprenaline was obtained in pre-contracted tracheal smooth muscle in the presence of three concentrations of aqueous-ethanolic extract, propranolol, and saline. Values of EC50 (the effective concentration of isoprenaline, causing 50% of maximum response) and dose ratio-1(CR-1) were measured. This effect was tested innon-incubated tracheal smooth muscle (group 1) and incubated tissues with chlorpheniramine (group 2). Concentration-response curves to isoprenaline in the presence of two higher concentrations of the extract in group 1 and all three concentrations in group 2 showed leftward shifts compared to isoprenaline curves produced in the presence of saline in both groups. EC50 obtained in the presence of propranolol was significantly higher than that of saline in both groups of experiments (p<0.05 for both cases). However, the EC50 obtained in the presence of two higher concentrations of the extract in group 1 and lower concentration in group 2 were non-significantly but those obtained of medium and high extract concentrations in the group 2 were significantly (p<0.05 for both cases)lower than those of saline. The values of (CR-1) obtained in the presence of all concentrations of the extract in groups1 and 2 were significantly lower than that of propranolol (p<0.05 to p<0.001).The results indicated a stimulatory effect of the P. olerace extract on ß 2-adrenoceptors of tracheal smooth muscle.

Novel relaxant effects of RPL554 on guinea pig tracheal smooth muscle contractility.

We investigated the effectiveness of RPL554, a dual PDE3 and 4 enzyme inhibitor, on airway smooth muscle relaxation and compared it with that induced by salbutamol, ipratropium bromide, glycopyrrolate or their combination on bronchomotor tone induced by different spasmogenic agents. Guinea pig tracheal preparations were suspended under 1g tension in Krebs-Henseleit solution maintained at 37°C and aerated with 95% O2 /5% CO2 and incubated in the presence of indomethacin (5μM). Relaxation induced by cumulative concentrations of muscarinic receptor antagonists (ipratropium bromide or glycopyrrolate), β2 -adrenoceptor agonists (salbutamol or formoterol), PDE3 inhibitors (cilostamide, cilostazol or siguazodan) or a PDE4 inhibitor (roflumilast) was evaluated in comparison with RPL554. Maximal relaxation was calculated (% Emax papaverine) and expressed as mean±SEM. Bronchomotor tone induced by the various spasmogens was reduced by the different bronchodilators to varying degrees. RPL554 (10-300μM) caused near maximum relaxation irrespective of the spasmogen examined, whereas the efficacy of the other relaxant agents varied according to the contractile stimulus used. During the evaluation of potential synergistic interactions between bronchodilators, RPL554 proved superior to salbutamol when either was combined with muscarinic receptor antagonists. RPL554 produced near maximal relaxation of highly contracted respiratory smooth muscle and provided additional relaxation compared with that produced by other clinically used bronchodilator drugs. This suggests that RPL554 has the potential to produce additional beneficial bronchodilation over and above that of maximal clinical doses of standard bronchodilators in highly constricted airways of patients.

Tumor Necrosis Factor Alpha Inhibits L-Type Ca(2+) Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway.

Tumor necrosis factor alpha (TNF-α) is a potent proinflammatory cytokine that plays a significant role in the pathogenesis of asthma by inducing hyperresponsiveness and airway remodeling. TNF-α diminishes the L-type voltage dependent Ca2+ channel (L-VDCC) current in cardiac myocytes, an observation that seems paradoxical. In guinea pig sensitized tracheas KCl responses were lower than in control tissues. Serum from sensitized animals (Ser-S) induced the same phenomenon. In tracheal myocytes from nonsensitized (NS) and sensitized (S) guinea pigs, an L-VDCC current (ICa) was observed and diminished by Ser-S. The same decrease was detected in NS myocytes incubated with TNF-α, pointing out that this cytokine might be present in Ser-S. We observed that a small-molecule inhibitor of TNF-α (SMI-TNF) and a TNF-α receptor 1 (TNFR1) antagonist (WP9QY) reversed ICa decrease induced by Ser-S in NS myocytes, confirming the former hypothesis. U0126 (a blocker of ERK 1/2 kinase) also reverted the decrease in ICa. Neither cycloheximide (a protein synthesis inhibitor) nor actinomycin D (a transcription inhibitor) showed any effect on the TNF-α-induced ICa reduction. We found that CaV1.2 and CaV1.3 mRNA and proteins were expressed in tracheal myocytes and that sensitization did not modify them. In cardiac myocytes, ERK 1/2 phosphorylates two sites of the L-VDCC, augmenting or decreasing ICa; we postulate that, in guinea pig tracheal smooth muscle, TNF-α diminishes ICa probably by phosphorylating the L-VDCC site that reduces its activity through the ERK1/2 MAP kinase pathway.

The Inhibitory Effects of Ferula assafoetida on Muscarinic Receptors of Guinea-Pig Tracheal Smooth Muscle

Background: In traditional medicine, asafoetida obtained from the roots of different Ferula assafoetida (F. asafoetida) is used for various therapeutic purposes such as whooping cough, pneumonia and bronchitis in children, as well as asthma. Objectives: In this study, the effect of asafoetida on muscarinic receptors of tracheal smooth muscle, as one possible mechanism responsible for the relaxant effect seen for the plant, was evaluated. Materials and Methods: The effects of three cumulative concentrations of aqueous extract of F. asafoetida (2.5, 5 and 10 mg/mL), 10 nM atropine, and saline on muscarinic receptors were tested in tracheal smooth muscle samples by performing cumulative concentration response curve to methacholine (a muscarinic receptor agonist) and assessing the shift in concentration response curves due to different concentrations of the extract and atropine. Results: The EC 50 obtained in the presence of higher concentration of the extract muscle was significantly higher compared to saline (P < 0.01). The maximum responses to methacholine in the presence of higher concentration of the extract (10 mg/mL) was significantly lower than that of saline (P < 0.05). The values of CR-1, obtained in the presence of all concentrations of the extract, were significantly lower compared to atropine in the experimental group (P < 0.01 to P < 0.001). Conclusions: These results showed an inhibitory effect for the extract of asafetida on muscarinic receptors of tracheal smooth muscle.

17β-Aminoestrogens induce guinea pig airway smooth muscle hyperresponsiveness through L-type Ca2+ channels activation

Therapy with estrogens is frequently used in menopausal women and as hormonal contraception. Because of its thrombotic effects, long term estrogen administration used in hormonal replacement therapy (HRT) and contraception could represent a health hazard. In this regard, 17β-aminoestrogens such as aminoestrol, butolame and pentolame have shown promising HRT potential, because they have a weak agonist estrogenic action and antithrombotic activity. Additionally, estrogens play a protective role in airway smooth muscle, but the effect of 17β-aminoestrogens on the airway smooth muscle has not been tested yet. In guinea pig tracheal smooth muscle pentolame and butolame induced hyperresponsiveness to histamine (His), carbachol (Cch) and KCl. Interestingly, aminoestrol did not show this effect at the highest concentration studied, it even lowered the contraction induced by Cch. The hyperresponsiveness induced by pentolame to His was abolished by nifedipine. In single tracheal myocytes, KCl induced an increment in the intracellular Ca2+ concentration [Ca2+]i, pentolame also showed an increase in [Ca2+]i and the addition of KCl in the plateau of this rise further significantly augmented the [Ca2+]i response. Additionally, in patch clamp experiments pentolame increased the L-type Ca2+ currents. Thus, 17β-aminoestrogens such as pentolame and butolame, but not aminoestrol, activate L-type Ca2+ channel to induced hyperresponsiveness to Cch, His and KCl in guinea pig tracheal smooth muscle. Due to its lack of effect on airways and to its anticoagulant characteristics, aminoestrol seems to be the best alternative in the HRT among the 17β-aminoestrogens studied.

Contribution of water and lipid soluble substances in the relaxant effects of Tymus vulgaris extract on guinea pig tracheal smooth muscle in vitro.

To examine the relaxant effects of hydro-ethanolic, macerated aqueous (MA) and lipidfree macerated aqueous (LFMA) extract of Tymus vulgaris on tracheal chains of guinea pigs. The relaxant effects of five cumulative concentrations of each extract (0.4, 0.8, 1.2, 1.6 and 2.0 g/100 mL) were compared with saline as negative control and five cumulative concentrations of theophylline (0.1, 0.2, 0.4, 0.6 and 0.8 mmol/L) on precontracted tracheal smooth muscle of guinea pig with 60 mmol/L KCl (group 1) and 10 µmol/L methacholine (group 2, n=6 for each group). In group 1 all concentrations of theophylline, three higher concentrations of hydro-ethanolic, two concentrations of LFMA and last concentration of MA extracts showed significant relaxant effects compared with that of saline (P<0.05 or P<0.01). Two lower concentrations of LFMA and all concentrations of MA except higher one caused contraction compared with saline (P<0.05 or 0.01). In group 2 experiments, all concentrations of theophylline, hydro-ethanolic, MA and LFMA extracts showed significant relaxant effects compared to that of saline (P<0.05 or P<0.01). In both groups, the relaxant effect of all concentrations of hydro-ethanolic extract were significantly higher than most concentrations of others (P<0.05 or P<0.01). The relaxant effect of different concentrations of three extracts were significantly greater in group 2 compared with group 1 experiments (all P<0.01). There were significantly positive correlations between the relaxant effects and concentrations for theophylline and all extracts in both groups (P<0.05 or P<0.01). Hydro-ethanolic extract has a potent weaker relaxant effect for other extracts from Tymus vulgaris on tracheal chains of guinea pigs.

Antihistamine Effect of a Pure Bioactive Compound Isolated from Slug (Diplosolenodes occidentalis) Material.

Folklore claims of the therapeutic effect of garden slug (Diplosolenodes occidentalis) extract used to relieve bronchoconstriction in asthmatic individuals were never validated scientifically. The aim of this study was to isolate the pure bioactive compound from slug extract causing this effect. The crude ground material was prepared in ethanol and after filtration, separation by flash column chromatography method was done. The structure was elucidated by data from hydrogen and carbon nuclear magnetic resonance (NMR) profiles. The bioactive compound was assessed for dose-dependent response effects on guinea pig tracheal smooth muscle pre-contracted with histamine. Receptor specificity studies were done by using HTMT dimaleate (H1 agonist). The type of antagonism was also identified. The pure component isolated from garden slug material was identified by spectral studies as glyceryl trilinolenate (GT). It caused dose-dependent relaxation in guinea pig tracheal smooth muscle strips pre-contracted with histamine, it acted via H1 type receptors and showed non-competitive antagonism. Glyceryl trilinolenate produced dose-dependent relaxation in tracheal smooth muscle strips in the presence of the agonist histamine. Glyceryl trilinolenate displayed non-competitive antagonism at H1 receptors in the trachea. This agent was able to alleviate bronchoconstriction in individuals presenting with atopic asthma in rural agricultural areas in Jamaica (verbal communications). It is possible that GT can be useful therapeutically to produce tracheal smooth muscle relaxation in individuals presenting with atopic asthma.

Relaxant effects of asafoetida extract and its constituent umbelliprenin on guinea-pig tracheal smooth muscle.

To investigate the relaxant effects of the asafoetida (Ferula Asafoetida Oleo-Gum-Resin) and its coumarin constituent umbelliprenin on tracheal chains of guinea pigs. The relaxant effects of three cumulative concentrations of the aqueous extract (2, 5 and 10 mg/mL), umbelliprenin (0.04, 0.2 and 0.4 mg/mL), theophylline (0.05, 0.1 and 0.15 mg/mL) and saline were examined by their relaxant effects on precontracted tracheal chains of guinea pig by 60 mmol/L KCl (group 1), and 10 μ mol/L methacholine (group 2). In group 1 all concentrations of theophylline and the highest concentration of the extract showed signififi cant relaxant effects compared with that of saline (P<0.01 for theophylline and P<0.05 for extract). In group 2, relaxant effects of all concentrations of theophylline, extract and two higher concentrations of umbelliprenin differed significantly compared with saline [P<0.01 for all cases except low concentration of umbelliprenin (0.04 mg/mL)]. There were no signififi cant differences between relaxant effects of the extract and theophylline in group 2. The relaxant effect of the extract in group 2 was signififi cantly greater than that of group 1 (P<0.01). The relaxant effect of the extract was significantly more potent than umbelliprenin in all groups (P<0.05 or P<0.01). These results showed a potent relaxant effect for the asafoetida extract on tracheal smooth muscle which is, at least, in part, due to its constituent umbelliprenin. A muscarinic receptor blockade was also suggested for the extract.