Contraction Of Tracheal Smooth Muscle Research Articles

Effect of antioxidants on airway smooth muscle contraction: action of lipoic acid and some of its novel derivatives on guinea pig tracheal smooth muscle

The aim of this study was to investigate the ability of (a) antioxidants, some related to alpha-lipoic acid (LA), (b) histone deacetylase (HDAC) inhibitors, and (c) hybrid compounds possessing both alpha-lipoic acid-derived antioxidant properties and HDAC inhibitory activity to inhibit guinea pig smooth muscle contraction. Guinea pig isolated tracheal rings (GPTR) were prepared and their isometric tension measured using a transducer. Histamine, carbachol and 5-hydroxytryptamine (5-HT) served as agonists. Tests with antigen (ovalbumin) used GPTR from sensitised guinea pigs or rings from non-sensitised animals that had been incubated for at least 2 h with diluted serum from sensitised animals. All antioxidants tested showed a relaxant effect on resting tension and on tension induced by histamine or carbachol, with EC(50)(s) of 0.2-5.0 mM and a rank order of potency: LA derivatives > glutathione (GSH) > ascorbic acid (AA). However, low concentrations (<50 microM) of GSH, AA and LA potentiated histamine-induced contractions. The benzamide HDAC inhibitor MGCD0103 inhibited mast cell activation and GPTR contraction produced by antigen and certain agonists, although a 2-6 h pre-incubation was required for those effects to be apparent. Two LA-benzamide HDAC hybrid compounds, UCL M084 and UCL M109 inhibited GPTR contraction after 30 min pre-incubation; however, even after long pre-incubation (up to 6 h) those hybrid compounds showed less potent inhibition of agonist-induced contraction than did MGCD0103. The results showed that GSH more potently inhibited contraction induced by histamine than that induced by 5-HT or carbachol, whereas LA, and especially UCL M084 and UCL M109, more potently blocked contraction induced by carbachol and 5-HT than that induced by histamine. For GSH, and possibly also for LA-type compounds, the inhibition of agonist-induced tracheal smooth muscle contractions may be due to NO formation. This study did not detect a synergistic relaxant effect in two compounds incorporating the structural union of a benzamide HDAC inhibitor terminus with a LA-derived antioxidant moiety.

Effects of bambuterol and terbutaline on isolated rat’s tracheal smooth muscle

Terbutaline (Bricanyl) and its prodrug Bambuterol (Bambec) are highly potent beta(2)-adrenoceptor agonists often used in asthma patients. Terbutaline in the form of inhaled short-acting bronchodilator relieves asthmatic symptoms. However, the effects of bambuterol given intratracheally have rarely been explored. To verify the effect of bambuterol and terbutaline, which act on the tracheal smooth muscle directly in vitro, we used our preparation to test the effects of bambuterol on isolated rat's tracheal smooth muscle compared with terbutaline. The following assessments of bambuterol and terbutaline were performed: (1) effect on tracheal smooth muscle resting tension; (2) effect on contraction caused by 10(-6) M methacholine as a parasympathetic mimetic; (3) effect of the drugs on electrically induced tracheal smooth muscle contractions. The results indicated that adding bambuterol induced a significant further contraction to 10(-6) M methacholine-induced contraction when the preparation was increased to 10(-4) M. But terbutaline elicited a relaxation response at a dose of 10(-6) M or more. Both bambuterol and terbutaline could inhibit electrical field stimulation (EFS) induced spike contraction. Terbutaline had an anti-cholinergic effect that could relieve asthmatic symptoms. But the effect of bambuterol acting on tracheal smooth muscle directly was controversial.

Effects of Flavin7 on allergen induced hyperreactivity of airways

Some studies have suggested that the polyphenolic compounds might reduce the occurrence of asthma symptoms. The aim of our experiments was to evaluate the effects of 21 days of the flavonoid Flavin7 administration on experimentally induced airway inflammation in ovalbumin-sensitized guinea pigs. We assessed tracheal smooth muscle reactivity by an in vitro muscle-strip method; changes in airway resistance by an in vivo plethysmographic method; histological picture of tracheal tissue; and the levels of interleukin 4 (IL-4), and interleukin 5 (IL-5) in bronchoalveolar lavage fluid (BALF). Histological investigation of tracheal tissue and the concentrations of the inflammatory cytokines IL-4 and IL-5 in BALF were used as indices of airway inflammation. Administration of Flavin7 caused a significant decrease of specific airway resistance after histamine nebulization and a decline in tracheal smooth muscle contraction amplitude in response to bronchoconstricting mediators. Flavin7 minimized the degree of inflammation estimated on the basis of eosinophil calculation and IL-4 and IL-5 concentrations. In conclusion, administration of Flavin7 showed bronchodilating and anti-inflammatory effects on allergen-induced airway inflammation.

Effects of lidocaine on rat’s isolated tracheal smooth muscle

Lidocaine has been used as an injection or spray solution to protect spasm reaction during tracheal intubation and bronchoscopy. The effect of lidocaine for local anesthesia is well known. However, the effect of the drug to tracheal smooth muscle is not well explored. We used our preparation to test the effectiveness of lidocaine on rat's isolated tracheal smooth muscle. The following assessments of lidocaine were performed: (1) effect on tracheal smooth muscle resting tension; (2) effect on contraction caused by 5 x 10(-6) M methacholine as a parasympathetic mimetic; (3) effect of lidocaine on electrically induced tracheal smooth muscle contractions. The addition of lidocaine induced a relaxation response to methacholine-induced contraction. Low doses of the drug resulted in a slight decrease in contraction and higher doses relaxed the trachea much more quickly. At 10(-3) M of lidocaine, almost 51% of 5 x 10(-6) M methacholine-induced contraction was decreased. Using the same concentration, the drug also could totally inhibit the electrical field stimulation-induced spike contraction. Lidocaine elicited a negligible response to the resting tension of trachea as the concentration increased. The study indicated that lidocaine could cause bronchodilation not only from blocking parasympathetic tone, but also from directly antagonizing the effect of cholinergic receptors.

ROLE OF EXTRACELLULAR Na+, Ca2+‐ACTIVATED Cl‐ CHANNELS AND BK CHANNELS IN THE CONTRACTION OF Ca2+ STORE‐DEPLETED TRACHEAL SMOOTH MUSCLE

1. In the present study, we investigated the series of events involved in the contraction of tracheal smooth muscle induced by the re-addition of Ca(2+) in an in vitro experimental model in which Ca(2+) stores had been depleted and their refilling had been blocked by thapsigargin. 2. Mean (+/-SEM) contraction was diminished by: (i) inhibitors of store-operated calcium channels (SOCC), namely 100 micromol/L SKF-96365 and 100 micromol/L 1-(2-trifluoromethylphenyl) imidazole (to 66.3 +/- 4.4 and 41.3 +/- 5.2% of control, respectively); (ii) inhibitors of voltage-gated Ca(2+) channels Ca(V)1.2 channels, namely 1 micromol/L nifedipine and 10 micromol/L verapamil (to 86.2 +/- 3.4 and 76.9 +/- 5.9% of control, respectively); and (iii) 20 micromol/L niflumic acid, a non-selective inhibitor of Ca(2+)-dependent Cl(-) channels (to 41.1 +/- 9.8% of control). In contrast, contraction was increased 2.3-fold by 100 nmol/L iberiotoxin, a blocker of the large-conductance Ca(2+)-activated K(+) (BK) channels. 3. Furthermore, contraction was significantly inhibited when Na(+) in the bathing solution was replaced by N-methyl-D-glucamine (NMDG(+)) to 39.9 +/- 7.2% of control, but not when it was replaced by Li(+) (114.5 +/- 24.4% of control). In addition, when Na(+) had been replaced by NMDG(+), contractions were further inhibited by both nifedipine and niflumic acid (to 3.0 +/- 1.8 and 24.4 +/- 8.1% of control, respectively). Nifedipine also reduced contractions when Na(+) had been replaced by Li(+) (to 10.7 +/- 3.4% to control), the niflumic acid had no effect (116.0 +/- 4.5% of control). 4. In conclusion, the data of the present study demonstrate the roles of SOCC, BK channels and Ca(V)1.2 channels in the contractions induced by the re-addition of Ca(2+) to the solution bathing guinea-pig tracheal rings under conditions of Ca(2+)-depleted sarcoplasmic reticulum and inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase. The contractions were highly dependent on extracellular Na(+), suggesting a role for SOCC in mediating the Na(+) influx.

The mechanisms of ethanol‐induced contraction of tracheal smooth muscle

We have shown that ethanol induces contraction of pulmonary artery smooth muscle. Therefore, we hypothesized that ethanol induces contractile responses of tracheal smooth muscle and extra cellular Ca2+ plays an important role on these responses. In vitro force measurements were performed in tracheal smooth muscle strips obtained from adult ferrets (n=33). TSM strips were treated with different concentration of ethanol (46 ‐ 322 mM). In another set of experiments strips were incubated in Ca2+ free Krebs ‐Hensleit solution or a Ca2+ channel blocker ‐ nifedipine was used and then treated with ethanol. The involvement of nitric oxide (NO) was studied by inhibition of nitric oxide synthase (NOS) using Nω‐nitro‐L‐arginine methyl ester. The ethanol induced concentration‐dependent contraction of TSM. Prior treatment of tissues with a single dose of ethanol increased the contractile responses of TSM to acetylcholine (p&lt;0.05). Lack of Ca2+ in medium resulted with decrease of contractile force of the TSM likewise Ca2+ channel blocker. Inhibition of NOS significantly increased contractile responses (p&lt;0.001).These data demonstrate that ethanol induces contraction of TSM and it appears to play a critical role in enhancing cholinergically mediated contractile responses of TSM. Extra cellular Ca2+ plays an important role on these responses and NO release as a relaxant molecule counteracts the effects of ethanol on TSM.

Activation of Vinculin Induced by Cholinergic Stimulation Regulates Tension Development in Tracheal Smooth Muscle (TSM)

The adhesion protein vinculin functions in focal adhesions to link actin filaments to membrane adhesion junctions by binding to the integrin‐binding protein talin at its head domain (Vh) and to actin filaments at its tail domain (Vt). In the inactive (closed) state, Vh and Vt bind to each other, masking the binding sites for actin and talin; in the active (open) state, vinculin binding sites for talin and actin are exposed. We hypothesized that the contractile activation of TSM might regulate conformation of vinculin and thereby contribute to the regulation of tension development. Stimulation of TSM tissues with acetylcholine (ACh) increased the co‐immunoprecipitation of vinculin and talin from tissue extracts, and increased vinculin phosphorylation at tyr 1065, an indicator of vinculin activation. Recombinant Vh (EGFP‐Vh) expressed in TSM tissues competed with endogenous vinculin for binding to talin and inhibited vinculin tyr 1065 phosphorylation. Expression of EGFP‐Vh depressed TSM contraction to ACh by 60% and inhibited ACh‐induced actin polymerization. EGFP‐Vh did not affect myosin light chain phosphorylation. We conclude that vinculin is activated in TSM by ACh, and that vinculin regulates tension by promoting connections between actin filaments and talin‐integrin adhesion complexes and by facilitating the initiation of actin polymerization.Support: AHA 0820074Z, HL 29289, HL 074099

Effects of a selection of histone deacetylase inhibitors on antigen- and agonist(s)-induced tracheal smooth muscle contraction

Effects of a selection of histone deacetylase inhibitors on antigen- and agonist(s)-induced tracheal smooth muscle contraction

Effects of cetirizine on isolated rat’s tracheal smooth muscle

Cetirizine (Zytec) is often used as a histamine receptor-1 (H(1)) antagonist in rhinitis patients who are suffering from sneezing and rhinorrhea. This H(1) antagonist is used as an oral tablet or nasal spray solution. The effect of H(1) antagonist on nasal mucosa in vivo is well known; however, the effect of the drug on tracheal smooth muscle has been rarely explored. Therefore, during administration of the H(1) antagonist for nasal symptoms, it might also affect the trachea via oral intake or inhalation. We used our preparation to test the effectiveness of Zytec on isolated rat's tracheal smooth muscle. The following assessments of Zytec were performed: (1) effect on tracheal smooth muscle resting tension; (2) effect on contraction caused by 5 x 10(-6) M methacholine as a parasympathetic mimetic; (3) effect of the drug on electrically induced tracheal smooth muscle contractions. Results indicated that addition of a parasympathetic mimetic to the incubation medium caused the trachea to contract in a dose-dependent manner. Addition of Zytec at doses of 10(-5) M or above elicited a relaxation response to 5 x 10(-6) M methacholine-induced contraction. Zytec could inhibit electrical field stimulation induced spike contraction, and basal tension was increased at the same time. However, it alone had a minimal effect on the basal tension of trachea as the concentration increased. This study indicated that high concentrations of Zytec might actually inhibit parasympathetic function of the trachea.

Adenosine induces a cholinergic tracheal reflex contraction in guinea pigs in vivo via an adenosine A1receptor-dependent mechanism

Adenosine induces dyspnea, cough, and airways obstruction in asthma, a phenomenon that also occurs in various sensitized animal models in which a neuronal involvement has been implicated. Although adenosine has been suggested to activate cholinergic nerves, the precise mechanism has not been established. In the present study, the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) induced a cholinergic reflex, causing tracheal smooth muscle contraction that was significantly inhibited by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 100 microg/kg) (P < 0.05) in anesthetized animals. Furthermore, the adenosine A(2) agonist 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680) induced a small reflex, whereas the A(3) selective agonist N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine (IB-MECA) was without effect. The tracheal reflex induced by CPA was also inhibited by recurrent nerve ligation or muscarinic receptor blockade (P < 0.001), indicating that a cholinergic neuronal mechanism of action accounted for this response. The cholinergic reflex in response to aerosolized CPA was significantly greater in passively sensitized compared with naive guinea pigs (P < 0.01). Chronic capsaicin treatment, which inhibited sensory nerve function, failed to inhibit CPA-induced reflex tracheal contractions in passively sensitized guinea pigs, although the local anesthetic lidocaine inhibited CPA-induced tracheal contractions. The effects of CPA on the reflex response was not dependent on the release of histamine from tissue mast cells or endogenous prostaglandins as shown by the lack of effect of the histamine H(1) receptor antagonist pyrilamine (1 mg/kg) or the cyclooxygenase inhibitor meclofenamic acid (3 mg/kg), respectively. In conclusion, activation of pulmonary adenosine A(1) receptors can stimulate cholinergic reflexes, and these reflexes are increased in allergic guinea pigs.

S‐CARBOXYMETHYLCYSTEINE INHIBITS CARBACHOL‐INDUCED CONSTRICTION OF EPITHELIUM‐DENUDED RAT AND HUMAN AIRWAY PREPARATIONS

1. The effects of s-carboxymethyl-L-cysteine (S-CMC), either administered orally to rats or incubated with tissue preparations from rats and humans, on isometric contractions of tracheal smooth muscle were investigated in the present study using an improved in vitro model of tracheal tube or ring preparations. The involvement of the tracheal epithelium in the observed effects was also investigated. 2. The experimental model permitted selective perfusion of the airway tube, luminal-IN or serosal-OUT, and measurement of airway smooth muscle contraction or relaxation in preparations with (+) or without (-) epithelium (Ep), excluding direct effects of airway mucus. 3. We found that oral pretreatment of rats with S-CMC (mixed with water; 200 mg/kg per day for 2 weeks), but not short pre-incubation of preparations in vitro (10(-3) mol/L S-CMC for 1 h), diminished the sensitivity of -Ep preparations to carbachol compared with controls (EC(50) (-log(10) mol/L) values: 5.5 +/- 0.1 vs 5.8 +/- 0.1, respectively, for IN perfusion (P < 0.005); 5.6 +/- 0.1 vs 5.9 +/- 0.1, respectively, for OUT perfusion (P < 0.005)), whereas the sensitivity of preparations to aminophylline was not affected. Normal sensitivity to carbachol stimulation was re-established if preparations were pre-incubated with capsaicin. 4. It was also found that longer pre-incubation (4 h) of ring-preparations of human bronchus with S-CMC (10(-5) mol/L) in vitro resulted in a diminished response to carbachol stimulation. 5. In conclusion, S-CMC had small inhibitory effects on the sensitivity of rat and human airway smooth muscle to carbachol, particularly in endothelium-denuded preparations. Whether the epithelium was responding to S-CMC by producing some contracting factor(s) requires further investigation.

Verapamil Induces Calcium Influx in the Trachea

Verapamil, a Ca2+entry blocker, can induce bronchorelaxation and bronchoconstriction. The mechanism of verapamil-induced bronchoconstriction is poorly understood. The present study determines the direct effect of verapamil on smooth muscle of isolated ovine airways and analyzes the mechanisms involved. Isolated tracheal strips were suspended in organ baths containing Krebs solution for isometric tension recording. Tissue responses to verapamil as assessed by basal tone were examined in the presence or absence of epithelium. The effects of verapamil on carbachol and cooling-induced contraction were also recorded. Measurement of unidirectional fluxes was carried out using 45Ca2+in the absence or presence of verapamil. Verapamil induced contractions of basal tracheal smooth muscle that were proportional to its concentrations. Removal of epithelium did not affect the verapamil contractile effect. Verapamil-induced contractions were abolished in Ca2+-free Krebs solution containing 2 mM EGTA. Verapamil increased the 45Ca2+influx into the tracheal smooth muscle. It caused relaxation of the muscle tone induced by carbachol or KCl, but it potentiated the effect of cooling-induced contraction. Verapamil induced Ca2+influx that may lead to bronchoconstriction. These results proved that verapamil may worsen bronchoconstriction; therefore verapamil should be used with caution in asthmatic individuals.

Effects of oxymetazoline on isolated rat’s tracheal smooth muscle

Oxymetazoline is often used as a decongestant in rhinitis patients who are suffering from nasal obstruction. It is used as a nasal drop or spray solution. The effect on nasal mucosa in vitro or in vivo is well known. However, the effect of the drug on tracheal smooth muscle has rarely been explored. During administration of the drug to the nose, it might affect the trachea via inhalation. We used our preparation to test the effectiveness of oxymetazoline on isolated rat's tracheal smooth muscle. A 5 mm long portion of rat trachea was submersed in 30 ml Kreb's solution in a muscle bath at 37 degrees C. Changes in tracheal contractility in response to the application of parasympathetic mimetic agents were measured using a transducer connected to a Pentium III computer equipped with polygraphy software. The following assessments were performed: (1) effect on tracheal smooth muscle resting tension; (2) effect on contraction caused by 10(-6)M methacholine as a parasympathetic mimetic; (3) effect of oxymetazoline on electrically induced tracheal smooth muscle contractions. Addition of parasympathetic mimetics to the incubation medium caused the trachea to contract in a dose-dependent manner. Addition of oxymetazoline induced a significant relaxation response when the preparation was up to 10(-4) M. At the same concentration, the drug also could inhibit EFS induced spike contraction. Oxymetazoline had negligible effect on the basal tension of trachea as the concentration increased. The degree of drug-induced tracheal contraction or relaxation was dose-dependent. The study indicated that high concentrations of oxymetazoline might actually antagonize cholinergic receptors of the trachea.

Red wine polyphenolic compounds inhibit tracheal smooth muscle contraction during allergen-induced hyperreactivity of the airways

The aims of the study were to investigate the short and long-term effects of Provinol (red wine polyphenolic compounds) on tracheal smooth muscle reactivity using an in-vitro model of ovalbumin-induced airway inflammation in guinea-pig trachea, and to evaluate the role of nitric oxide (NO) in the bronchodilatory effect of Provinol. The amplitude of tracheal smooth muscle contraction in response to mediators of bronchoconstriction - histamine (10 nM-1 mM), acetylcholine (10 nM-1 mM) and to allergen (ovalbumin 10(-5)-10(-3) g mL(-1)) was used as a parameter of tracheal smooth muscle reactivity. To test the short-term effects of Provinol, isolated tracheal strips were pre-treated for 30 min with Provinol (10(-4) mg mL(-1)) alone or in combination with Nomega-nitro-L-arginine methyl ester (L-NAME; 10(-6) mol L(-1)). To test the long-term effects of Provinol, isolated tracheal strips were prepared from guinea pigs that had been treated for 14 days with Provinol (20 mg kg(-1) per day) alone or in combination with L-NAME (40 mg kg(-1) per day). Incubation of tracheal smooth muscle with Provinol decreased the amplitude of contraction in response to ovalbumin, histamine and acetylcholine. The non-selective NO synthase inhibitor L-NAME partially abolished the effect of Provinol on acetylcholine and ovalbumin-induced but not histamine-induced bronchoconstriction. A similar profile was observed after 14 days' oral administration of Provinol. In conclusion, Provinol inhibited the allergen- and spasmogen-induced contraction of tracheal smooth muscle in ovalbumin-sensitized guinea pigs via a mechanism that was mediated at least partially through the metabolism of NO.

The Extract from Nandina domestica THUNBERG Inhibits Histamine- and Serotonin-Induced Contraction in Isolated Guinea Pig Trachea

Although the fruit of Nandina domestica THUNBERG (ND) has been used to treat respiratory disorders such as coughing and breathing difficulty in Japan for many years, very little is known about mechanisms underlying its action. In the present study, we investigated effects of the crude extract from ND (NDE) and one of its constituents, nantenine, on contractile responses in isolated guinea pig tracheal ring preparations. In normal experimental condition, guinea pig trachea remained tonically contracted during the resting state, and addition of NDE (1 mg/ml) caused a relaxation of tracheal smooth muscles, but had little effect on the responsiveness of trachea to acetylcholine. The basal, tonic contraction was abolished by the presence of atropine and indomethacin. In this condition, NDE at 0.1-1 mg/ml inhibited histamine-induced contraction in both competitive and non-competitive manners. NDE at 0.01-1 mg/ml inhibited serotonin-induced contraction in a competitive manner. Nantenine (2-20 microM) did not affect histamine-induced contraction, and slightly inhibited serotonin-induced contraction. These results suggest that NDE has inhibitory effects on tracheal smooth muscle contraction, and nantenine cannot account solely for this effect of NDE.

Mechanism underlying H 2O 2-induced inhibition of acetylcholine-induced contraction in rabbit tracheal smooth muscle

The mechanism underlying the inhibition by H 2O 2 of acetylcholine-induced contraction was investigated in epithelium-denuded strips of rabbit trachea. Acetylcholine (10 μM) generated a phasic, followed by a tonic increase in both the intracellular Ca 2+ concentration ([Ca 2+] i) and force. Although the acetylcholine-induced tonic contraction was around 9 times the high K +(80 mM)-induced one, the two stimulants induced similar [Ca 2+] i increases (around 0.2 μM), indicating that acetylcholine generates tonic contraction via increases in both [Ca 2+] i and myofilament Ca 2+-sensitivity. H 2O 2 (30 μM) (a) enhanced the acetylcholine-induced tonic (not phasic) increase in [Ca 2+] i but attenuated both phases of the acetylcholine-induced contraction and (b) enhanced the high K +-induced increase in [Ca 2+] i but did not modify the high K +-induced contraction. In β-escin-skinned strips, application of acetylcholine in the presence of GTP enhanced the contraction induced by 0.3 μM Ca 2+ so that its amplitude became similar to that induced by 1 μM Ca 2+. H 2O 2 (30 μM) attenuated the contraction induced by 0.3 μM Ca 2+ (alone or in the presence of acetylcholine) but not those induced by higher concentrations of Ca 2+ alone (0.5 μM and 1 μM). These results indicate that H 2O 2 acts directly on contractile proteins in rabbit tracheal smooth muscle to inhibit the contraction induced by low concentrations of Ca 2+ (< 0.5 μM). An action of H 2O 2 that increases [Ca 2+] i (and thereby masks this reactive-oxygen-induced inhibition of myofilament Ca 2+-sensitivity) is apparent in the presence of high K + but not of acetylcholine. Thus, in rabbit tracheal smooth muscle H 2O 2 downregulates myofilament Ca 2+-sensitivity more potently during acetylcholine-induced contraction than during high-K +-induced contraction, leading to an effective inhibition of the former contraction.

Essential oil of Pterodon polygalaeflorus inhibits electromechanical coupling on rat isolated trachea

The present work studied the effects of the essential oil of Pterodon polygalaeflorus (EOPP), a plant used to treat bronchitis and amigdalytis, on rat airway smooth muscle in vitro. In Ca 2+-containing medium, EOPP (100–1300 μg/ml) inhibited preferentially high KCl- than 5-HT-induced muscle contractions in a concentration-dependent fashion, but did affect neither basal muscle tension nor ACh-induced contractions. In preparations maintained in either 60 mM K + or 10 μM ACh in Ca 2+-free medium, EOPP (100, 600 and 1300 μg/ml) inhibited maximum contractile response induced by cumulative Ca 2+ addition (0.1–20 mM). Verapamil (10, 30 and 100 μg/ml), a Ca 2+ channel blocker, also inhibited Ca 2+-induced concentration–effect curve in presence of ACh in Ca 2+-free medium, whilst it was ineffective to decrease cholinergic contractions in Ca 2+-containing medium. In presence of 150 mM K + in Ca 2+-containing medium, EOPP (1300 μg/ml) did not reversed ACh-induced contractions. In contrast, under similar conditions, EOPP almost fully relaxed cholinergic contractions of tracheal smooth muscle in Ba 2+-containing medium. In medium containing 10 mM tetraethylammonium and 2 mM Ba 2+ instead of Ca 2+, both EOPP (1300 μg/ml) and verapamil (∼5 μg/ml) significantly decreased ACh-induced contractions. Thus, in rat isolated trachea, EOPP induces inhibitor effects on contractions preferentially triggered by an electromechanical coupling mode.

Dynamic association between α‐actinin and β‐integrin regulates contraction of canine tracheal smooth muscle

The adhesion junctions of smooth muscle cells may be dynamically regulated during smooth muscle contraction, and this dynamic regulation may be important for the development of active tension. In the present study, the role of alpha-actinin during smooth muscle contraction was evaluated in tracheal smooth muscle tissues and freshly dissociated cells. Stimulation with acetylcholine (ACh) increased the localization of alpha-actinin at the membrane of freshly dissociated smooth muscle cells, and increased the amount of beta1 integrin that coprecipitated with alpha-actinin from muscle tissue homogenates. GFP-alpha-actinin fusion proteins were expressed in muscle tissues and visualized in live freshly dissociated cells. GFP-alpha-actinin translocated to the membrane within seconds of stimulation of the cells with ACh. Expression of the integrin-binding rod domain of alpha-actinin in smooth muscle tissues depressed active contraction in response to ACh. Expression of the alpha-actinin rod domain also inhibited the translocation of endogenous alpha-actinin to the membrane, and inhibited the association of endogenous alpha-actinin with beta1-integrin in alpha-actinin immunoprecipitates from tissue extracts. However, the expression of alpha-actinin rod domain peptides did not inhibit increases in myosin light chain phosphorylation or actin polymerization in response to stimulation with ACh. Results suggest that contractile stimulation of smooth muscle causes the rapid recruitment of alpha-actinin to beta-integrin complexes at the membrane, and that the recruitment of alpha-actinin to integrin complexes is necessary for active tension development in smooth muscle.

Differential Rho‐kinase dependency of full and partial muscarinic receptor agonists in airway smooth muscle contraction

In airway smooth muscle (ASM), full and partial muscarinic receptor agonists have been described to have large differences in their ability to induce signal transduction, including Ca2+-mobilization. Despite these differences, partial agonists are capable of inducing a submaximal to maximal ASM contraction. To further elucidate transductional differences between full and partial muscarinic receptor agonists, we investigated the contribution of Rho-kinase (an important regulator of Ca2+-sensitization) to methacholine-, pilocarpine- and McN-A-343-induced bovine tracheal smooth muscle (BTSM) contraction, using the selective Rho-kinase inhibitor Y-27632. In addition, we measured Ca2+-mobilization and -influx in BTSM cells in response to these agonists in the absence and presence of Y-27632. Whereas treatment with Y-27632 (1 microM) significantly decreased potency (pEC50) for all agonists, maximal contraction (Emax) was reduced by 23.4+/-2.8 and 50.4+/-7.9% for the partial agonists pilocarpine and McN-A-343, respectively, but was unaffected for the full agonist methacholine. However, Emax of methacholine became Rho-kinase dependent after taking away its receptor reserve using the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard. Pilocarpine and McN-A-343 induced a very small Ca2+-mobilization and -influx as compared to methacholine. In addition, an inverse relationship of these two parameters with the Rho-kinase dependency was observed. Interestingly, no inhibitory effects of Y-27632 were observed on Ca2+-mobilization and-influx for all three agonists, indicating that the effects of Y-27632 on contraction are most likely on the level of Ca2+-sensitization. In conclusion, in contrast to the full agonist methacholine, the partial muscarinic receptor agonists pilocarpine and McN-A-343 are dependent on Rho-kinase for their maximal contractile effects, presumably as a consequence of differences in transductional reserve, indicating an agonist-dependent role for Rho-kinase in ASM contraction. Moreover, an inverse relationship exists between Rho-kinase dependency and both Ca2+-mobilization and Ca2+-influx for these agonists.

The Effects of Lidocaine, Bupivacaine or Combined with Enflurane on the Contraction of Tracheal Smooth Muscle in Guinea Pig

Background: The effects of local and inhalation anesthesia on the contraction of the tracheal smooth muscle have been reported. However, when inhalation and local anesthesia are administered simultaneously, their effects on the contraction of the airway smooth muscle are not well known. Therefore, this study evaluated the effects of lidocaine, bupivacaine, with or without enflurane, on the histamine induced tracheal smooth muscle contraction in guinea pigs. Methods: Isolated tracheal rings of guinea pigs were suspended in Krebs solution. The contractions were recorded isometrically using a transducer. A contraction was induced by histamine ( M). Enflurane (1 MAC) was administered for 15 min after administering the histamine. The cumulative dose responses of the lidocaine and bupivacaine ( M, M, M, M, M, and M), enflurane-lidocaine and enflurane-bupivacaine groups were checked. Results: The contraction by histamine were inhibited by lidocaine ( M and M) and bupivacaine ( M, M and M). In addition, the contractions were inhibited more significantly in the bupivacaine group ( M, M, M, M and M) after administering enflurane. Conclusions: Lidocaine and bupivacaine inhibited the contractions induced by histamine. The contractions by bupivacaine were further inhibited after administering enflurane.