Preparation of bovine tracheal smooth muscle for in vitro pharmacological studies

doi:10.1016/1056-8719(95)00043-H

Journal of Pharmacological and Toxicological Methods

Volume 34, Issue 2, October 1995, Pages 103-108

https://doi.org/10.1016/1056-8719(95)00043-H Get rights and content

Abstract

In this study a new method is described for standardization of both length and weight of bovine tracheal smooth muscle strips to be used for measuring airway responsiveness in vitro. Tracheal smooth muscle was cut into strips 2.5-mm thick and 10-mm long with an average wet weight of 25.9 ± 0.6 mg (n = 5). By use of preloads varying from 0.5 to 10 g, it was found that 5 g caused the highest contraction and relaxation of the tissues in response to pharmacological agents. Moreover, at a preload of 5 g the variations among the maximal responses induced by histamine, methacholine, KCl, and salbutamol were very small (standard errors being 6.8%, 5.0%, 2.5%, and 1.6% of the corresponding average maximal response) as well as were the variations in the pD₂ values.

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Cited by (7)

Role of β-adrenoceptors, cAMP phosphodiesterase and external Ca <sup>2+</sup>on polyamine-induced relaxation in isolated bovine tracheal strips
2010, Pharmacological Reports
Citation Excerpt :
Bovine tracheal smooth muscle strips were obtained from healthy male cattle (10–13 months old, breed –Asturiana de los Valles, Asturias, Spain) [5]. Four to six adjacent transversal, similar rectangular segments (approximately 15 x 2.5 mm and 155.14 ± 3.31 mg in weight) were obtained and mounted in a 6-ml organ bath containing Krebs solution (118 mM NaCl; 4.75 mM KCl; 2.5 mM CaCl2; 1.19 mM KH2PO4;25 mM NaHCO3; 1.2 mM MgSO4; and 11 mM glucose) according to a previously described and validated method [5, 16]. Tissues were allowed to stabilize for at least 2 h, and the buffer solution was renewed every 30 min.
Polyamines relax several smooth muscles and elicit cardiotonic effects in the rat heart via interactions with β-adrenoceptors. The aim of this work was to establish whether β₂-adrenoceptors were involved in polyamine-relaxation of bovine tracheal strips. Endogenous polyamines displaced the specific radioligand, [ H]dihydroalprenolol, but spermine was the most potent. The polyamines elicited an acute transient relaxation, which was independent of β-adrenoceptor activation, followed by a maintained component, which was shown to be dependent on β-adrenoceptor activation because it was antagonized and reversed by propranolol. Polyamines did not alter salbutamol-induced acute relaxation. Polyamines modified the salbutamol-induced long-term effect on airway tone, which was shown by a partial reversal of β-adrenoceptor desensitization. This process was delayed by α-difluoromethylornithine, but spermine increased the latency and time of reversal and decreased receptor desensitization. Putrescine prolonged the time-constant without changes in the desensitization. Spermine, but not putrescine, might block Ca²⁺ channels, because it relaxed KCl- or electrical stimulated-contractions, which are related to Ca²⁺ influx, and the inhibition of cAMP phosphodiesterase activity. These differences might explain the functional differences observed between putrescine and spermine. Therefore, polyamines may modulate airway smooth muscle tone and interfere with the mechanism of receptor desensitization via several mechanisms involving β₂-adrenoceptors, Ca²⁺ influx and cAMP phosphodiesterase.
Modulatory role of endogenous androgens on airway smooth muscle tone in isolated guinea-pig and bovine trachea; involvement of β<inf>2</inf>-adrenoceptors, the polyamine system and external calcium
2008, European Journal of Pharmacology
Androgens relax several smooth muscles, including the airways. They also contract ileum and myocardium via nongenomic mechanisms. To find out whether androgens modulate airway smooth muscles in different species and further assess their mechanism of action, regarding the role of β-adrenoceptors, polyamines and extracellular Ca²⁺, and the modulation of contraction, 5α-dihydrotestosterone, testosterone and 5β-dihydrotestosterone were used. A preliminary study was performed to evaluate the effect of 5α-dihydrotestosterone, a non-aromatisable derivate of testosterone, in isolated guinea-pig trachea and a more exhaustive characterisation was followed in bovine trachea, to also characterise the effect of testosterone and 5β-dihydrotestosterone. The androgens elicited a nongenomic epithelium-independent relaxation of the trachea which had been precontracted. In the bovine trachea, the order of potency was: testosterone > 5α-dihydrotestosterone = 5β-dihydrotestosterone. This effect was inversely proportional to the magnitude of carbachol-raised tone and was independent of β₂-adrenoceptors, since the β-blockers, propranolol and ICI-118,551, and β₂-adrenoceptor desensitisation did not modify 5α-dihydrotestosterone-elicited relaxation. 5α-Dihydrotestosterone was unable to displace the radiolabel, [³H]dihydroalprenolol, from these receptors in the binding assay. Polyamine synthesis was not involved in this androgen effect, since an ornithine decarboxylase inhibitor, α-difluoromethylornithine, was ineffective. The androgens were more effective relaxing bovine trachea precontracted by KCl (80 mM), suggesting a calcium entry blockade, as reported for several smooth muscles. This mechanism might be involved in the observed 5α-dihydrotestosterone facilitation of salbutamol-relaxation. Androgens facilitated carbachol-elicited contraction independently of polyamine synthesis, contrary to what has been reported in the ileum. Therefore, androgens modulate tracheal smooth muscle tone which might be of importance in the regulation of airway reactivity.
The protective role of epithelium-derived nitric oxide in isolated bovine trachea
2002, Pulmonary Pharmacology and Therapeutics
Airway epithelial cells from bovine airways can release relaxant factors such as nitric oxide (NO) and prostaglandin E₂ and the removal of airway epithelium results in an increased responsiveness of smooth muscle to spasmogen stimuli. In this study, we assessed whether or not epithelial NO modulates the contractile response of bovine trachea in vitro.
Cumulative concentration–response curves to acetylcholine (ACh), histamine (Hist) and 5-hydroxytryptamine (5-HT) were obtained in both intact and epithelium denuded tracheal strips in the presence of indomethacin (10 μM).
In intact, but not in epithelium denuded strips, preincubation with the NO synthase inhibitor L-N^{^G}-Nitro-arginine methyl ester (L-NAME), but not with D-NAME, shifted to the left the concentration–response curve to ACh (pD₂ values in the absence and in the presence of L-NAME were 3.47±0.1 and 4.60±0.1, respectively; P<0.05) and to Hist (pD₂ in the absence and in the presence of L-NAME: 3.89±0.1 and 4.54±0.1, respectively; P<0.05). This effect was reversed by L-arginine (1 mM), but not by D-arginine. The contractile response to 5-HT was not affected by L-NAME in either intact or epithelium denuded strips.
These data suggest that NO is an epithelial relaxant factor modulating airway cholinergic and histaminergic contraction of bovine trachea and that the activation of the epithelial NO synthase is a mediator-specific process.
Effects of Drugs on Air Ways
2015, Drug Discovery and Evaluation: Pharmacological Assay, Fourth Edition
Sex hormones modulate salbutamol-elicited long-term relaxation in isolated bovine tracheal strips
2011, Pharmacology
M<inf>2</inf> muscarinic receptors inhibit forskolin- but not isoproterenol- mediated relaxation in bovine tracheal smooth muscle
1998, Journal of Pharmacology and Experimental Therapeutics

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Original articlePreparation of bovine tracheal smooth muscle for in vitro pharmacological studies

Abstract

Pulm Pharmacol

J Pharmacol Methods

J Allergy

Eur J Pharmacol

Relationship between smooth muscle volume and contractile response in airway tissue. Isometric versus isotonic measurement

J Pharmacol Exp Ther

The effect of airway epithelium on smooth muscle contraction in bovine trachea

Br J Pharmacol

Airway reactivity in ponies with recurrent airway obstruction (heaves)

J Appl Physiol

Original article
Preparation of bovine tracheal smooth muscle for in vitro pharmacological studies