ACh-release was evaluated from the outflow of [
3H]. The validity of this method for measuring [
3H]-ACh has been demonstrated by Kilbinger et al. in 1991 [
3]. The strength of this method is that it does not require cholinesterase inhibitors, thus avoiding unphysiological high concentrations of ACh resulting in M-receptor auto-inhibition. A potential problem of this technique is that the release of [
3H]-ACh may not reflect accurately the release of endogenous ACh [
18], but this has not been confirmed [
19]. In order to measure quantitatively the electrically induced ACh-release, neuronal re-uptake of [
3H]-choline was blocked by hemicholinium-3, resulting in [
3H]-ACh depletion in postganglionic cholinergic nerve endings with each consecutive electric stimulation. The number of consecutive stimulations in each study was therefore limited to three. However, [
3H]-ACh-release is affected by the magnitude of depletion with previous stimulations. If [
3H]-ACh-release is altered by study drugs, depletion can either be increased or reduced with the second stimulation, thus leaving less or more [
3H]-ACh available to be released with the third electrical stimulation. In the present study there were no significant differences in [
3H]-ACh-release after second stimulation between control muscles and muscles treated with either GLY 10
−9 M, IND 10
−8 M, IND 10
−6 M, or with GLY 10
−9 M co-incubated with IND 10
−8 M. With the highest GLY concentrations, the [
3H]-ACh-release was greater than control, therefore there is a possibility that [
3H]-ACh-release after GLY 10
−6 M incubation might have been underestimated.
The frequency of 4 Hz for the assessment of electrically-induced [
3H]-ACh-release and contractile response was chosen from frequency-response curves, where it caused a near half-maximal contraction. The concentrations of GLY and IND used in the electrically-induced [
3H]-ACh-release and contractile response experiments were chosen from the frequency- and exogenous ACh-response curves characterizing GLY antagonism and from previous data with other β-adrenoceptor agonists [
8], respectively.
Electrically-induced ACh-release may be attenuated in isolated bovine trachealis by stimulation of pre-junctional β
2-adrenoceptor [
8]. Thus, in experiments with GLY alone, propranolol was added. Activation of muscarinic receptors stimulates synthesis and release of prostaglandins, which in turn reduces ACh-release [
4,
20,
21]. Thus, to avoid any confounding effect of prostaglandins, studies were done in the presence of indomethacin.
The present study was done using bovine trachealis. Therefore, any extrapolation of our data to human bronchi either in healthy subjects or in patients having COPD, with different β
2-adrenoceptor and M-receptor density [
22], should be draw cautiously. The choice of this tissue was based on the sufficient availability to conduct the large number of experiments necessary for the study and because muscarinic autoregulation [
6] is present in this tissue similar to human airways [
1,
2,
5].
Isometric contraction does not represent what happens in vivo, where airway smooth muscle contraction occurs under auxotonic conditions. However, this does not invalidate conclusions regarding interactions between drugs acting on different receptors.
Long-acting β
2-adrenoceptor agonists and cholinergic M-receptor antagonists are recommended by expert committees [
23] and guidelines [
24,
25] as the cornerstone treatment for COPD. No specific recommendations are given on the strategy to optimize the use of these drugs. One option is starting treatment with either one of them and, if not sufficient to control symptoms, increasing the dosage of the same drug or adding a second one; an alternative option is starting with low-dosage combination treatment. The M-receptor antagonist ipratropium caused a bronchodilator effect that was maximal in patients with bronchitis but not in those with bronchial asthma [
26]. Based on this finding, it has long been thought that cholinergic tone and the amplifying effect of airway wall thickening are the main mechanisms for airway narrowing in COPD [
26‐
29]. Because no difference was observed between ipratropium and a combination of fenoterol plus theophylline, the choice between β
2-adrenoceptor agonists and M-receptor antagonists as monotherapy for COPD has been considered as a matter of side effects more than efficacy. If these data from isolated bovine airways can be extrapolated to human airways, then the results would suggest that muscarinic antagonists might represent the treatment of choice and the better strategy for monotherapy in COPD.
The superiority of GLY over IND in attenuating airway smooth muscle contraction might appear surprising owing to the expected opposing effects of these drugs on ACh-release from pre-junctional post-ganglionic nerves. As other available anti-muscarinic drugs, GLY is not selective for M
3-receptor [
30,
31], but it increases ACh-release by antagonizing also pre-junctional M
2 and/or M
4 receptors [
3,
5,
6]. By contrast, β
2-adrenoceptor agonists may reduce ACh-release by opening pre-junctional Ca
2+-dependent K
+ channels [
8,
32]. In the present study, at concentration of GLY less than 10
−7 M, electrically-induced ACh-release remained unaltered, whereas at concentrations of 10
−7 M and 10
−6 M the ACh-release was increased. This difference in response might be due to blocking ganglionic M
1-receptors at low concentrations offsetting the effect on post-ganglionic M
2-receptor at low but not high concentrations [
33‐
35]. The increments of ACh-release with GLY or GLY + IND were on average between 20 and 27%. Based on a previous study using opioid agonists [
7] and frequency-response curves (Additional file
1: Figure S1), we estimated that these changes would have resulted in changes of force between 8 and 10% in the absence of drugs acting at post-junctional level. These changes, however, cannot be quantitatively extrapolated to in vivo conditions for a series of reasons. First, the relationship between ACh-release and force is not linear and the frequency of vagal firing may be variably affected by different stimuli [
36]. Second, changes in airway smooth muscle force translate into changes of linear length depending on opposing loads [
27]. Finally, changes in linear length translate into changes in airway caliber depending on airway geometry [
29]. It can be speculated that changes in force may translate into greater changes of airway caliber in COPD than healthy subjects because of the increased thickness of airway walls and the loss of lung elastic recoil [
27‐
29].
Interestingly, the contractile responses were abolished by 10
−7 M and 10
−6 M concentrations of GLY, despite an increase of ACh-release. Presumably, complete blockade of post-junctional M
3-receptor prevented a contractile response to the increased ACh-release. This is consistent with the absence of contractile non-cholinergic stimuli in bovine trachealis [
6]. Conversely, at 10
−5 M IND reduced ACh-release and, importantly, the net inhibitory effects on contractile responses were less than with GLY lower concentrations.
A major objective of the present study was to investigate whether the GLY-IND combination may have additive or more-than additive effects in reducing airway smooth muscle contraction and ACh-release. The combination of GLY 10
−8 M with IND 10
−7 M attenuated contractions more than GLY 10
−8 M, or IND 10
−7 M given separately, and the expected based on Bliss independent criterion [
14,
15]. This more than additive effect in conjunction with the insignificant effects of IND 10
−7 M alone on ACh-release suggest a synergistic effect at post-junctional level, rather than a functional antagonism at pre-junctional level.
M
2-receptors are expressed not only pre-junctionally on nerves but also post-junctionally on airway smooth muscle cell membrane [
37,
38]. Inhibition of post-junctional M
2 receptors by muscarinic antagonist inhibits the receptor-coupled G
iα subunit, thus favoring the adenylyl cyclase activity, the cAMP accumulation, and thereby the relaxant effect of β
2-adrenoceptor agonists [
39,
40]. Therefore, non-selective muscarinic antagonists may increase the relaxing effects of β
2-adrenoceptor agonists. Indeed, in human bronchi a greater cAMP concentration has been observed with a GLY-IND combination than with IND alone [
11].
An important finding of this study is that adding a low concentration (10−8 M) of GLY to IND 10−7 M had an inhibitory effect on airway smooth muscle contraction that was superior to increasing IND concentration by 10 times and not inferior to increasing it by 100 times.