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Clinical and Experimental Pharmacology & Physiology
 
Clin Exp Pharmacol Physiol. 2017 July; 44(7): 795–802.
Published online 2017 June 28. doi:  10.1111/1440-1681.12763
PMCID: PMC5519946

Effects of presynaptic muscarinic cholinoreceptor blockade on neuromuscular transmission as assessed by the train‐of‐four and the tetanic fade response to rocuronium

Summary

This study investigated the effect of muscarinic M1 and M2 receptor antagonists on the rocuronium‐induced train of four (TOF) fade and tetanic fade, respectively. Ex‐vivo phrenic nerves and diaphragms were obtained from adult Sprague‐Dawley rats and stabilized in Krebs buffer; the nerve‐stimulated muscle TOF fade was observed at 20 s intervals. For the TOF study, phrenic nerves and diaphragms were incubated with pirenzepine (an M1 blocker) at concentrations of 0 nmol L−1 (control), 10 nmol L−1 (PZP10), or 100 nmol L−1 (PZP100). Rocuronium was then administered incrementally until the first twitch tension had depressed by >95% during TOF stimulation. The mean TOF ratios were compared when the first twitch tensions were depressed by 40%‐50%. For the tetanic fade study, 50 Hz/5 s tetani was applied initially, 30 min after the administration of a loading dose of rocuronium and methoctramine (an M2 receptor blocker, loaded at 0 μmol L−1 [control], 1 μmol L−1 [MET1], or 10 μmol L−1 [MET10]). The EC 95 of rocuronium was significantly lower in the PZP10 group than in the control group. In the PZP10 group, the TOF ratios at 50% and first twitch tension depression were significantly lower than those in the control group (P=.02). During tetanic stimulation, the tetanic fade was significantly enhanced in the MET10 group compared to the other groups. This study shows that antagonists of muscarinic M1 and M2 receptors affect the rocuronium‐induced neuromuscular block as demonstrated by the reduced EC 95 and TOF ratios (M1 antagonist, pirenzepine) or the enhanced 50‐Hz tetanic fade (M2 antagonist, methoctramine).

Keywords: acetylcholine release, methoctramine, muscarinic cholinoreceptor, neuromuscular physiology, pirenzepine, rocuronium

1. INTRODUCTION

Several types of receptors are found at neuromuscular presynaptic sites.1, 2 Some of these receptors function together to control the release of acetylcholine (ACh) during rest or stimulation.2 For example, muscarinic and purinergic receptors interact at presynaptic neuronal sides. Of these receptors, muscarinic facilitatory M1 and inhibitory M2 receptors regulate the fine‐tuning actions of ACh release on neuronal firing, and these receptors are in turn regulated by purinergic receptors.3, 4, 5, 6

Train‐of‐four (TOF) stimulation is performed by indirect stimulation at 2 Hz four times and enables the monitoring of recovery from neuromuscular blockade.7 The TOF ratio (TOFR) is an important measure of the margin of safety of neurotransmission during the perioperative period as well as of muscle function in the presence of muscle diseases, such as myasthenia gravis. As a TOF assessment uses low‐frequency stimulation, we hypothesized that the muscarinic M1 receptors may be primarily involved in the release of ACh via TOF stimulation because the M1 receptor function is evoked predominantly via low‐frequency (<5 Hz) stimulation upon ACh release at the neuromuscular presynaptic side.4 We further hypothesized that if the function of the M1 receptor is inhibited by an M1 blocking drug, such as pirenzepine (PZP), the TOFR may be modulated.

Tetanic stimulation provides useful information about the depth of blockade during deep neuromuscular paralysis produced by non‐depolarizing muscle relaxants,8 and it reflects the ability to maintain function during repetitive nerve stimulation (eg exercise). Tetanic stimulation is used in clinical practice at 50 Hz for 5 s, during which time tetanic tension is sustained and does not fade in normal (non‐diseased) muscles. In contrast, when the neuromuscular block is produced by non‐depolarizing neuromuscular‐blocking drugs, the muscle tension is not sustained, and fade is detected. The post‐tetanic count, the phenomenon involving post‐tetanic facilitation of twitch tensions after tetanic stimulation, is used to assess the depth of recovery (or paralysis) from deep neuromuscular block when there is no response to TOF stimulation.9, 10 Fade during tetanic stimulation is considered as a presynaptic event. During rest and low‐frequency stimulation (<5 Hz), facilitatory muscarinic M1 receptor function is predominant. However, the transmitter adenosine, which is co‐released with ACh during nerve stimulation at the synapses, is markedly increased when the stimulation frequency is increased to 50 Hz; it eventually reaches levels capable of activating the facilitatory adenosine A2A receptors, which counteract M1 receptors, and potentiates M2 inhibitory receptors.4 Therefore, we hypothesized that presynaptic M2 receptors may play an important role when high‐frequency stimulation (50‐Hz tetanic stimulation) is applied during post‐tetanic count monitoring.

The nicotinic ACh receptor is primarily postsynaptically involved in neuromuscular signal transduction, and the muscarinic cholinoreceptor is one of the neuromuscular presynaptic modulating receptors. As such, the modulating effects of the M1 and M2 muscarinic cholinoreceptor on neurotransmission were assessed by decreasing the margin of safety through the administration of rocuronium. Accordingly, in the present study, we primarily aimed to identify the changes in variables induced during neuromuscular blockade by rocuronium after neuromuscular presynaptic muscarinic M1 or M2 receptors in an ex‐vivo phrenic nerve/diaphragm model were blocked; we then sought to compare these changes in the presence of different concentrations of muscarinic cholinoreceptor blocking agents. Therefore, we investigated whether PZP dihydrochloride contributes to TOF fade triggered by rocuronium, a muscle relaxant, and assessed the EC95 of rocuronium. Furthermore, we assessed whether methoctramine (MET) hydrate contributes to tetanic fade triggered by a 50‐Hz tetanic stimulation after a small dose of rocuronium.

2. RESULTS

2.1. Effects of the PZP‐induced muscarinic M1 receptor block on TOFR and EC95 of rocuronium

Each experimental group consisted of eight phrenic nerve/hemidiaphragm tissue specimens, and a total of 24 rat diaphragms were used. Each preparation was randomly allocated to treatment with rocuronium alone (control) or to one of two PZP concentrations (10 nmol L−1 [PZP10] or 100 nmol L−1 [PZP100]) followed by rocuronium. The concentration of rocuronium required to cause a >95% depression in the first twitch tension of TOF response (T1) in the PZP10 group was significantly lower than that in the control group (mean values, 37.42 and 42.10 μmol L−1, respectively; P=.024) (Table 1).

Table 1

Ex‐vivo analysis of T1 depression and train‐of‐four ratio

The concentration‐response curves of rocuronium for T1 depression and the TOFR in the PZP10 group were shifted to the left, as compared with those in the control group (Figure 1). The mean EC95 and EC50 values for rocuronium in each group are shown in Table 1. The following equation was derived for T1 depression: y=50 + 50sin(Ωx), where y represents T1 depression, x represents the rocuronium concentration, and Ω represents the steepness of the curve. The Ω of PZP10 was larger than that of the control and PZP100 groups (P=.001 and .012, respectively) (Figure 1C, Table 1). The following equation was derived for TOFR: y=1 − λx 2, where y represents the TOFR and x represents the rocuronium concentration. The λ of the PZP10 group was significantly larger than that of the other groups (P=.001) (Figure 1D, Table 1), but no significant difference was observed between the Ω and λ values of the control and PZP100 groups (P=.39 for T1 depression, P=.83 for TOFR). At T1‐response depression of 50%, the TOF ratio was significantly lower in the PZP10 group than in the control group (0.251 ± 0.13 vs 0.364 ± 0.121; P=.02) (Figure 2). However, no significant differences were found between the PZP100 group and the control group for any of the variables examined.

Figure 1

Actions of rocuronium on rat phrenic nerve/hemidiaphragm tissues. (A) Dose‐response curves of the effect of rocuronium on T1 depression and (B) train‐of‐four ratio (TOFR) values in the control, PZP10, and PZP100 groups. The R2 ...

Figure 2

TOFR values at 40%‐50% of T1 twitch tension depression. The TOFR value in the PZP10 group was significantly lower than that in the control group (*P=.02). However, the corresponding value in the PZP100 group did not differ significantly from that ...

2.2. Effect of muscarinic M2 receptor blockade by MET on tetanic fade

A total of 30 phrenic nerve/hemidiaphragm tissue preparations were obtained, and each tissue specimen was randomly allocated to treatment with one of three concentrations of MET (0 μmol L−1 [control, n=10], 1 μmol L−1 [MET1, n=10], or 10 μmol L−1 [MET10, n=10]) with rocuronium. No overall changes in T1 twitch tension or in TOFR during the initial period (ie before administration of rocuronium), pre‐incubation period (ie before administration of MET), or post‐incubation period (ie 30 min after administration of MET) with different concentrations of MET were observed. The first tetanic twitch tensions (A values, illustrated in the inset figure in Figure 4B) of the control, MET1, and MET10 groups during the initial and pre‐incubation periods did not exhibit any significant changes. The A values during the post‐incubation period were significantly lower (P=.01) than the initial values of the initial tetanic stimulation when compared within the group (Table 2), but there were no significant differences between the groups. The last tetanic twitch tension depression (B values, illustrated in inset figure in Figure 4B) and the change of the tetanic fade ratios became significant after the administration of rocuronium. The values during tetanic stimulation are shown in Table 2.

Table 2

Parameters obtained via tetanic stimulation

When comparing the R value (which is the ratio of B over A) within the group, we found significant changes during the post‐incubation period in all the groups in comparison with the initial R values within each group; however, only the post‐incubation R values of the MET10 group were significantly lower than those of the control or MET1 groups (P=.005) (Table 2, Figure 3A). The percentage changes in R values (%R=100×R post‐incubation/R pre‐incubation) were larger in the MET10 group than in the other groups (P=.005) (Figure 3B).

Figure 3

(A) Comparison of the changes in the tetanic fade ratio after incubation with MET (R post‐incubation/R pre‐incubation). During the initial and pre‐incubation (Pre) periods, tetanic fade ratios did not significantly differ between ...

3. DISCUSSION

In this ex‐vivo rat phrenic nerve/hemidiaphragm study, we found that presynaptic muscarinic cholinoreceptors play a role in modulating nerve‐stimulated neuromuscular response, particularly during the administration of rocuronium. In particular, neuromuscular presynaptic M1 receptors were found to play an important role in the rocuronium‐induced decrease in twitch tension and TOF fade. The EC50 and EC95 values of rocuronium and the TOFR at ~50% depression of the T1 were significantly lower in the PZP10 group than in the control group. These findings suggest that the facilitatory muscarinic M1 receptor is functional when the phrenic nerve is subjected to TOF stimulation; however, the findings obtained from the PZP100 group did not suggest this.

Pirenzepine is generally used as a muscarinic M1‐selective blocking agent because its affinity for M1 receptors is ~200 times more than for M2 receptors,11 which indicates that it is not absolutely M1‐specific. Atropine acts as a non‐specific muscarinic blocker to modulate ACh release by inhibiting both M1 and M2 receptors, which decreases spontaneous miniature end plate potential (mEPP) frequency and increases evoked ACh release.2, 12 By modulating the spontaneous quantal release, presynaptic M1 and M2 receptors preserve and stabilize synaptic function.12 TOF fade during neuromuscular blockade is a well‐known presynaptic phenomenon that is primarily mediated by the nicotinic ACh receptor (nAChR).13, 14, 15, 16, 17, 18 However, in an in‐vivo rat study, it was found that the dihydro β erythroidine (DHβE)‐induced blockade of the presynaptic α3β2 nAChR did not cause any change in twitch tension or TOF fade.19 We speculate that, in the PZP100 group, all these factors resulting from the partial M2 blocking effect of PZP may have influenced the results and led to reduced differences in T1 tension depression and TOF fade.

This study also indicated that the muscarinic M2 receptor plays an important role in tetanic fade during tetanic stimulation at 50 Hz for 5 s. The variables obtained during 50‐Hz tetanic stimulation were influenced when M2 receptors were blocked by MET. Other authors have used MET at 1 μmol L−1 and reported some effects on ACh release in similar experiments.4, 6, 20, 21 However, we did not observe any definitive changes at this concentration in the present study. Instead, we observed some unexpected changes by increasing the MET concentration to 10 μmol L−1.

In the present study, because rocuronium was administered before MET, some postsynaptic nAChRs were preoccupied by rocuronium, and thus, the responses to ACh at the postsynaptic sides may have been more dependent on the amount of ACh. In such an environment, the rapid depletion of ACh due to the blockade of the M2 receptors and the lack of accumulation of ACh at the synaptic junction could result in the failure to maintain twitch tension, making the tetanic fade more prominent in turn. In another study on frog end‐plates,22 blockade of M2 muscarinic ACh receptor by MET provoked a rise of quantal release and the lengthening of late releases, but a different study did not find this lengthening.23 This discrepancy between study findings indicates that this is a species‐specific phenomenon. Another possible reason for this discrepancy was that the MET had some open channel‐blocking action. Bixel et al. showed that polymethylenetetramines such as MET can bind to the transmembrane M2 domain within the ion channel of nAChRs as non‐competitive inhibitors.24 In their study, the MET as a non‐competitive inhibitor had an IC50 of 4.2 μmol L−1. In the present study, the concentration of MET in the MET10 group was more than twice the IC50 of MET on nAChR, and therefore, postsynaptic nAChR may have been affected by MET. But the nAChR used in Bixel's experiment was comprised of the two α subunits and the β, γ, and δ subunits24—that is, it was the ‘immature’ form of nAChR. Moreover, in our pilot study conducted without rocuronium, no changes in A, B, and R values were obtained by tetanic stimulation in the presence of 10‐μmol L−1 MET. These findings suggest MET has a minimal effect on postsynaptic nAChR.

The present study had several limitations. First, this study was conducted in an ex‐vivo environment, wherein the pharmacokinetic properties of each antagonist were excluded. It might be difficult to investigate this result in an in‐vivo study because M1 and M2 mAChRs are also present in the brain, autonomic nervous system, and heart. Therefore, additional data are required to support the extension of the current results to the fine‐tuned in‐vivo conditions.

Second, this study was of a functional nature, and the amounts of ACh at the neuromuscular junction may have differed during each indirect stimulation among the three groups. Similar experiments on presynaptic muscarinic and coupled serine threonine kinases have been performed by other authors who explained the function of presynaptic mAChRs in ACh release under various conditions,19, 20, 21 but most of these studies used different recording methods and muscles. These studies also reported that neuromuscular presynaptic mAChRs may have an overall conservative effect of presynaptic junctions by limiting neurotransmission. As such, we did not assess the molecular differences between control, PZP10, and PZP100 tissue samples via chemical analysis. Instead, we obtained twitch tensions using the rat hemidiaphragm by indirectly stimulating the phrenic nerve. If the difference in the number of released ACh molecules was insufficient to yield differences in postsynaptic nAChR activation, no significant change in twitch tension was observed via the externally evoked indirect supramaximal stimulation. According to the pharmacology of neuromuscular blocking agents, muscle twitch tension is maintained until sufficient numbers of postsynaptic nAChRs have been blocked by neuromuscular anatogonists.25, 26 This so‐called ‘margin of safety’ concept describes the relationship between neuromuscular antagonists, ACh, and nAChRs. We found that T1 depression was rapidly achieved, and TOF fade was exaggerated at a relatively low PZP concentration of 10 nmol L−1, particularly in comparison to the control group.

The third limitation of this investigation arose from the fact that several types of receptors (eg mAChRs and nAChRs) are also responsible for presynaptic ACh release at the neuromuscular junction. Muscarinic cholinoreceptors may have some role postsynaptically as well, but in the smooth muscle, developing muscles, and cultured muscle.27, 28 The ACh receptor described so far in skeletal muscle is nicotinic.29 In addition to the presynaptic muscarinic cholinoreceptors and nAChRs, presynaptic purinergic A1 and A2A receptors modulate the spontaneous and evoked release of ACh. When ACh is released from the presynaptic terminal by spontaneous or evoked stimulation, the concentration of the endogenous purinergic agonist, adenosine, increases at neuromuscular junctions and then controls ACh release through a feedback mechanism.30 Several studies have reported conflicting results regarding the effect of presynaptic purinergic receptor activation on ACh release. In one study, micromolar concentrations of adenosine reduced the evoked quantal release or spontaneous release of ACh,31, 32 but another report found that submicromolar concentrations exerted the opposite effect.32, 33 As such, we tried to apply the same frequency (2 Hz for TOF and 50 Hz for tetani) in all groups to ensure that the environment of adenosine for neuromuscular purinergic A1 and A2A at the neuromuscular junction was similar.

In conclusion, the overall function of neuromuscular presynaptic mAChRs is to preserve or save the presynaptic function in the presence of various neuronal stimulations. This function may be affected when the balance of action between these receptors is disrupted. Moreover, we noted that presynaptic muscarinic M1 receptors can modulate the rocuronium‐induced EC95 and TOF fade after PZP administration. Indirect stimulation at <5 Hz (the most commonly applied TOFR) is often used to monitor neuromuscular blockade, but the TOFR may be altered when the M1 receptor is blocked by PZP because the facilitatory effect of ACh release on repeated stimulation is inhibited. Our present study findings indicated that patients taking medications targeting the muscarinic M1 receptor (eg PZP) before or during surgery may exhibit modulations or distortions in the values that are representative of recovery from neuromuscular blockade. In addition, although the M2 receptor has an inhibitory effect on ACh release when activated, its antagonism by MET may waste the ACh released by evoked stimulation, in turn affecting the tetanic tension of the hemidiaphragm. As such, our findings emphasize that neuromuscular monitoring findings in patients treated with a muscarinic cholinoreceptor antagonist (eg PZP) must be interpreted with care.

4. METHODS

4.1. Basic study design and sample preparation

The study protocol was approved before study initiation by the Ethics Committee of the Laboratory of Animal Research, Asan Institute of Life Science (Seoul, Korea) on 1 December 2012 (protocol no. 2012‐14‐255, 2015‐14‐076). All animals were raised at a constant temperature of 22°C and maintained under a regular diurnal cycle with food and water supplied ad libitum. The size and weight of the phrenic nerve/hemidiaphragm tissue specimens were obtained, and the lengths of their base widths (diaphragm width attached to the thoracic wall) are described in Table 3. To ensure tissue viability throughout the experiments, tissue specimens were immersed in Krebs buffer solution (118 mmol L−1 NaCl, 2.5 mmol L−1 CaCl2, 4.7 mmol L−1 KCl, 1.2 mmol L−1 MgSO4, 1.4 mmol L−1 KH2PO4, 25 mmol L−1 NaHCO3, 11 mmol L−1 α‐D‐glucose) maintained at 35°C with 95% O2/5% CO2 with continuous bubbling. Pirenzepine and MET were purchased from Sigma‐Aldrich Korea (Gyeonggi Do, Republic of Korea). Two different PZP stock solutions and two different MET stock solutions were prepared and stored at 4°C in a refrigerator. These stock solutions were prepared such that the same volumes were used to achieve different concentrations of PZP or MET. The stock solutions were discarded 2 weeks after preparation.

Table 3

Characteristics of tissue specimen

4.2. TOF fade study protocol

Twenty‐four male Sprague‐Dawley rats, with an average weight of 223.02 ± 19.98 g (range, 196.59‐266.92 g), were used in the study. Each rat was intraperitoneally anaesthetized with 5‐μg/g thiopental sodium. The phrenic nerve/hemidiaphragm tissues were immediately obtained and fixed to frames with electrodes. The tissue specimens were then mounted in an organ bath and immersed in 75‐mL oxygenated Krebs buffer solution. The tendinous portion of the diaphragm of each specimen was attached to a Grass FT03 Force Transducer (Grass Instruments, West Warwick, RI, USA), and 40‐mN resting tension was applied. The phrenic nerve was attached to a bipolar electrode and stimulated using a Grass S88 Stimulator (Grass Instruments). Supramaximal stimulation with a square wave pulse of 0.2 ms was administered every 30 s at a TOF time of 2 Hz. All waveforms were acquired, displayed using the PowerLab 4/26 Data Acquisition System (AD Instruments, Sydney, NSW, Australia), and stored on an offline personal computer system using LabChart 7 software (AD Instruments, Colorado Springs, CO, USA).

Phrenic nerve/hemidiaphragm specimens were randomly allocated to one of three PZP groups: control group (PZP at 0 nmol L−1), PZP10 group (PZP at 10 nmol L−1), and PZP100 group (PZP at 100 nmol L−1). For each sample, twitch tension was serially monitored after a 30 min stabilization time. To ensure the designated concentration of PZP was correct in each group, the same volume of Krebs solution or two different PZP stock solutions were added to the organ bath. A 30 min reaction time was allowed in each group before the addition of a 400 μg loading dose of rocuronium (Esmeron®; MSD Korea, Seoul, Korea). Subsequently, a 200 μg booster dose of rocuronium was added to the organ bath. We used a 10 min cycle until >95% depression of the T1 was achieved. The study protocol is illustrated in Figure 4A.

Figure 4

Study protocol. (A) Diagram of the TOF study. The baseline stimulation pattern is the TOF stimulation, which was repeated every 20 s. (B) Diagram of the tetanic fade study. The baseline stimulation pattern is the TOF stimulation, which was repeated ...

4.3. Tetanic fade study protocol

Thirty male Sprague‐Dawley rats, with an average weight of 243.40 ± 43.84 g (range, 193.70‐325.44 g), were used in this study. Specimens were prepared as described in the TOF study protocol and allocated to one of three MET concentration groups: 0 μmol L−1 (control group), 1 μmol L−1 (MET1 group), and 10 μmol L−1 (MET10 group). After a 30 min stabilizing period, an initial 50‐Hz tetanic stimulation was administered for 5 s. A resting time of 30 min was then allowed, until the T1 tensions recovered to levels measured just before the tetanic stimulation (reference T1 tension). After it was confirmed that the T1 tension had recovered to >95% of the reference T1 tension, rocuronium loading doses were administered. A rocuronium loading dose of 100 μg was considered to be suitable for inducing tetanic fade without T1 tension depression or TOF fade. Approximately 20 min was allowed for sufficient reaction time of rocuronium. A second 50‐Hz tetanic stimulation was then administered for 5 s, and a resting time of 30 min was again permitted. After the T1 tensions returned to >95% of the reference T1 tension, MET was administered to achieve concentrations of 0, 1, or 10 μmol L−1 in the organ bath. After a reaction time of 30 min, the third (final) 50‐Hz tetanic stimulation was administered, and each tetanic fade parameter (A, B, and R, illustrated at the inset figure in Figure 4B) was measured and calculated. As a final step, a rocuronium dose‐response study was performed 30 min after the administration of the third tetanic stimulation. This study protocol is illustrated in Figure 4B.

4.4. Statistical analysis

The primary end‐point of this study was the concentration of rocuronium required to depress T1 by >95%. To achieve this goal, the rocuronium concentration was serially increased in Krebs buffer by repeatedly adding loading and booster doses of the drug at regular intervals while recording twitch tension. The second study end‐point was the mean TOFR when T1 depression was in the range of 50%‐60%. In the clinical setting, the recovery index (ie the period of T1 recovery from 25% to 75%) is useful for comparing recovery rates from certain neuromuscular blocking agents, including PZP. The concentration‐response relationships during this period are somewhat linear; therefore, the middle of this period was considered suitable for TOFR comparisons.

The results are expressed as means and standard deviations or means and standard errors. All doses were converted to μmol L−1. The group average TOFR value at the point where T1 twitch depression was in the range of 50%‐60% was estimated and is expressed as a mean and standard deviation. Graphs were plotted and statistical analyses were completed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). For T1 depression, we used the following equation: y=50 + 50sin(Ωx), where y represents the T1 depression, x represents the concentration of rocuronium, and Ω represents the slope of the regression curve (R 2=0.868). For TOFR, we used the following equation: y=1 − λx 2, where y represents TOFR, x represents the concentration of rocuronium, and λ represents the slope of the regression curve (R 2=0.832). The mean group values of Ω and λ were compared using the Kruskal‐Wallis test and Bonferroni's post‐hoc test. Rocuronium EC50 and EC95 values for twitch tension data were calculated by fitting nonlinear regression curves to group data. Statistical significance was accepted for P‐values <.05.

DISCLOSURE

The authors have no conflicts of interest to declare in association with this work.

ACKNOWLEDGEMENTS

These authors are members of the Neuromuscular Physiology Research Team at the Laboratory of Animal Research, Asan Institute of Life Science, Seoul, Korea.

Notes

Kim YB, Lee S, Lee KC, Kim HJ, Ro YJ, Yang H‐S. Effects of presynaptic muscarinic cholinoreceptor blockade on neuromuscular transmission as assessed by the train‐of‐four and the tetanic fade response to rocuronium. Clin Exp Pharmacol Physiol. 2017;44:795‐802. https://doi.org/10.1111/1440-1681.12763

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