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Comparative Study
. 2017 Jul 28;18(1):145.
doi: 10.1186/s12931-017-0627-5.

Interactions between glycopyrronium and indacaterol on cholinergic neurotransmission and contractile response in bovine trachealis

Michele Baroffio  1 , Giovanni Barisione  2 , Vito Brusasco  3
Affiliations
Comparative Study

Interactions between glycopyrronium and indacaterol on cholinergic neurotransmission and contractile response in bovine trachealis

Michele Baroffio et al. Respir Res. .

Abstract

Background: Muscarinic-receptor antagonists and β-adrenoceptor agonists are used, alone or in combination, as first-line treatment for chronic obstructive pulmonary disease. Both drugs decrease airway smooth muscle tone by post-junctional mechanisms but they may have opposing effects on pre-junctional acetylcholine (ACh)-release.

Methods: We studied the effects of the muscarinic-receptor antagonist glycopyrronium (GLY), the β-adrenoceptor agonist indacaterol (IND) and their combination on electrically-induced ACh-release and contractile response in isolated bovine trachealis. Data were analyzed by paired t-test and analysis of variance for repeated or independent measures with Newmann-Keuls post-hoc test when appropriate.

Results: GLY 10-8 M decreased contractile response by 19 ± 6% (p = 0.010) without altering ACh-release. GLY 10-7 M and 10-6 M almost abolished contractile responses even if the ACh-release was increased by 27 ± 19% (p < 0.001) and 20 ± 8% (p = 0.004), respectively. IND 10-7 M had no significant effects on contractile response and ACh-release, whereas IND 10-6 M reduced contractile response by 24 ± 12% (p = 0.002) without altering ACh-release. IND 10-5 M decreased contractile response by 51 ± 17% (p < 0.001) and ACh-release by 22 ± 11% (p = 0.004). Co-incubation with GLY 10-8 M and IND 10-7 M did not alter ACh-release but inhibited contractile response by 41 ± 8% (p < 0.001). The latter effect was greater than with GLY 10-8 M, or IND 10-7 M, or IND 10-6 M given separately (p < 0.001 for all). The increment of ACh-release caused by GLY was attenuated by IND 10-5 M, though this did not affect contractile response.

Conclusions: At equimolar concentration, GLY alone attenuates airway smooth muscle contraction more than IND, despite an increased ACh-release. Combination of GLY with IND at submaximal concentrations has more than additive effect suggesting a synergistic post-junctional effect. Adding GLY to IND provides a greater inhibitory effect on airway smooth muscle contraction than increasing IND concentration.

Keywords: Acetylcholine; Airway smooth muscle; Long-acting muscarinic antagonist; Long-acting β-adrenoceptor agonist.

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Conflict of interest statement

Ethics approval

Bovine tracheas were obtained, after permissions, from BLM Carni S.r.l., via Brughiera 10, 16030 Tribogna, Genoa and Fasce Gian Mario Ditta Individuale, via Recroso 241/bis, 16024 Lumarzo, Genoa. Ethical approval was not required because none of the authors was involved in the care or killing of the animals. Bovine care and slaughtering procedures were done according to the Italian law.

Consent for publication

Not applicable.

Competing interests

M.B. and G.B. have no competing interests to declare. V.B. has acted in a consultancy capacity and given lectures and has received fees from Boehringer Ingelheim, Dompé, GSK, Menarini, Novartis, AstraZeneca, Almirall. V.B. institution has received research support from Novartis and Chiesi.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Temporal sequences of studies performed to evaluate the effects of glycopyrronium (GLY) and indacaterol (IND) alone or in combination (*) on electrically-evoked contractile response and [3H]-acetylcholine (ACh)-release. Grey and black lines are referred to control and test muscles, respectively
Fig. 2
Fig. 2
Example of [3H]-ACh-release (a and b panels) and isometric force (c and d panels) in response to electrical stimulation in control (a and c panels) and test (b and d panels) bovine trachealis. DPM are disintegrations per min. S1, S2, and S3 represent first, second, and third electrical stimulation, respectively. Dashed lines represent the extrapolated spontaneous [3H]-ACh-release. The grey areas (A1, A2, and A3) are the [3H]-ACh-release induced by the corresponding stimulations. The electrically-induced [3H]-ACh-release decrease after each stimulation (A1 > A2 > A3) suggesting pre-junctional neuronal depletion of [3H]-ACh. ♦ represent radioactivity measurements of superfusate by liquid scintillation counting. Combination of GLY(○) and IND (×) inhibited the electrically-induced contractile response without altering the [3H]-ACh-release
Fig. 3
Fig. 3
a: frequency-response curves (FRC) obtained in 48 bovine trachealis from 6 animal in the absence or presence of different GLY concentrations. Data (means ± SD) are expressed as percent of 64-Hz-induced contraction obtained for each trachealis strip during the first FRC. b: Schild regression to exogenous ACh obtained on 56 bovine trachealis from 7 animals. Regression line was calculated by linear regression analysis of mean values (x) obtained in 7 different experiments. Dashed lines define the 95% confidence limits and arrow is referred to the GLY pA2 value. The dose ratio is the concentration of ACh inducing half-maximal contraction (EC50) in the presence of a given concentration of GLY divided for the EC50 for ACh in the absence of GLY. GLY inhibited both exogenous ACh- and electrically-evoked contractions
Fig. 4
Fig. 4
Effect of GLY (○, n = 13) and IND (×, n = 12) alone or combined (Δ, n=12) on electrically-induced [3H]-ACh-release and force (a and b panels, respectively). Continuous and interrupted lines indicate paired and unpaired data, respectively. Data are means ± SD; *, significantly different from zero (p < 0.05); #, significantly different from GLY 10−8 M (p < 0.001), IND 10−7 M, and IND 10−6 M (p < 0.001) alone
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