Interaction of agonists of a different subtype of the nAChR and carvacrol with GABA in Ascaris suum somatic muscle contractions

In our study, pyrantel caused concentration-dependent contractions of neuromuscular preparation of A. suum (Fig. 1A). After incubation with 3 μM of GABA, the EC₅₀ of pyrantel increased significantly from 8.44 to 28.22 nM (Table 1). However, when the muscle flaps were incubated with GABA 3 μM and carvacrol 100 μM at the same time, the EC₅₀ of pyrantel reached as much as 44 nM, which is significantly higher than the control value and the EC₅₀ of pyrantel after incubation with GABA 3 μM (Table 1). Part of this inhibitory effect is reversible, as after washing, the EC₅₀ value of pyrantel returned to the level observed in the presence of GABA 3 μM, but did not reach the initial control level. On the other hand, the value of the maximum contractile effect (R _max) of pyrantel has not changed in the presence of GABA 3 μM, but decreased significantly after incubation with combination of GABA and carvacrol (Table 2 and Fig. 1B).

Figure 1:

The control value of the bephenium EC₅₀ was 0.62 μM and it increased nonsignificantly to 0.96 μM in the presence of GABA 3 μM (Table 1). A representative recording of contractions caused by bephenium and the sigmoid concentration dose–responses plot are shown in Figure 2A, B. Furthermore, the EC₅₀ value of bephenium reached a level of 1.44 μM in the presence of GABA and carvacrol, which is highly significant compared both to the control and the EC₅₀ of bephenium obtained in the presence of GABA (Table 1). Something different was obtained with the maximum contractile effect of bephenium (R _max), because it did not change in the presence of GABA. However, the combination of GABA and carvacrol significantly reduced the R _max value from 2.2 to 0.89 g (Table 2).

Figure 2:

The EC₅₀ of morantel did not change significantly in the presence of GABA and in the presence of a combination of GABA and carvacrol (morantel EC₅₀ = 3.72, 5.69, and 7.94 nM) (Table 1). Moreover GABA 3 μM did not change the R _max of morantel either, but the combination of GABA and carvacrol significantly reduced the value of the maximal contractile effect (Table 2). Figure 3 shows a representative recording of A. suum muscle strip contractions caused by morantel (Fig. 3A) and sigmoid concentration dose–responses plot (Fig. 3B).

Figure 3:

The applied concentration of GABA (3 μM) did not cause relaxation of the tested muscle strips.

We believe that the concept of simultaneous use of compounds with multiple sites of antiparasitic action is able to prevent the development of resistance in parasites. We have shown in previous studies that carvacrol is most likely to act as a non-competitive inhibitor of nAChRs of nematodes (Marjanović et al., 2020; Trailović et al., 2015). It is known that one of the mechanisms for the development of resistance involves a change in the receptor to which the drug binds (Abongwa et al., 2017). For example, it is known that pyrantel resistance is associated with a modification of the target nicotinic receptor properties (Robertson et al., 2000). Whether this can also refer to the change of only certain subtypes of the nicotinic receptor has not been clarified.

Pyrantel is an anthelmintic from the tetrahydropyrimidine group. It is used in veterinary and human medicine against gastrointestinal nematodes. Pyrantel is a fairly selective agonist of nAChRs. It produces depolarization and contraction of the nematode body muscle which causes spastic paralysis. Pyrantel is a relatively potent agonist (EC₅₀ ~ 10 µM) on levamisole receptor subtypes (L-subtype) of nematode nAChRs. Our results are in accordance with the presented data. In our study, the contractile EC₅₀ of pyrantel for A. suum muscle flaps was 8.44 μM, and it increased more than threefold in the presence of GABA, without altering the R _max. Such an inhibition is manifested by competitive antagonists, but GABA certainly is not a competitive antagonist of nAChRs. We previously obtained a similar result with ACh and hypothesized that this phenomenon occurs as a consequence of the binding of GABA, in low concentrations, to the GABA receptor in the synapses between inhibitory interneurons and motoneurons (synaptic effect). This process immediately activates the competitive antagonistic excitatory mechanism through excitatory interneurons (Trailović et al., 2015). On the other hand, this study shows that the addition of carvacrol had a non-competitive antagonistic effect because the R _max was reduced by more than three times. We hypothesize that carvacrol cannot bind presynaptically but rather postsynaptically to a different site on nAChR than pyrantel, resulting in non-competitive antagonism.

Bephenium is an anthelmintic compound used against human and dog hookworms and gastrointestinal parasitic nematodes in sheep (Burrows, 1958). Bephenium activates the B-subtype of the nAChRs of nematodes (Martin and Robertson, 2007), but there is no more detailed information on the mechanism of action. In our study, GABA did not alter the EC₅₀ of bephenium, nor did it affect the maximal contractile effect (R _max). Furthermore, the addition of carvacrol led to a significant increase in bephenium EC₅₀ but also a decreased the R _max. This is obviously mostly non-competitive antagonism and can be explained by the fact that carvacrol binds to a different site on the receptor relative to the binding site of bephenium, and that the B-subtype of nAChRs are predominantly located postsynaptically.

The results obtained with morantel are especially interesting. Morantel also belongs to tetrahydropyrimidines, but it is still not clear which subtype of nAChRs it predominantly prefers. The incubation of the muscle strips with GABA did not significantly change the EC₅₀ of morantel, nor the R _max value. There was no significant change in EC₅₀ of morantel even after the incubation with the combination of GABA + carvacrol, but the R _max was significantly reduced. Similar to bephenium, we can state that the inhibitory effect of GABA is almost absent, while the combination of GABA and carvacrol exhibits mostly non-competitive antagonism. As before, we could assume that the receptors to which morantel binds are predominantly localized postsynaptically, which causes the absence of activation of inhibitory interneurons, and consequent competitive response. Courtot et al. (2015) reported that ACR-26 and ACR-27, the nAChRs subunits from Parascaris equorum expressed in Xenopus oocyte, are able to form a functional nAChR highly sensitive to pyrantel and morantel.

In our previous study (Trailović et al., 2015), GABA 3 μM shifted the acetylcholine EC₅₀ to the right, producing a dose-ratio (EC_50control/EC_50GABA) of 2.39, while the R _max was reduced by 30%. Now, after the incubation with 3 μM of GABA the dose ratios were 3.33 for pyrantel, 1.54 for bephenium, and 1.60 for morantel. On the other hand, R _max for pyrantel, bephenium, and morantel was reduced by 9, 0.1, and 14.6%, respectively. It is obvious that GABA most effectively inhibits the effect of pyrantel and that the majority of this effect is competitive. We assume that this is the result of the synaptic effect in the synapses between inhibitory interneurons and motoneurons. However, when GABA was combined with carvacrol the dose-ratio (EC_50control/EC_{50GABA + carvacrol}) was 6.93, 2.25, and 2.13 for pyrantel, bephenium, and morantel, respectively, while R _max decreased by 70, 60, and 65%, respectively. Their effect is more dominantly non-competitive, but also indicates that the combined use of GABA receptor agonists and nicotinic receptor antagonists can effectively inhibit the neuromuscular system of nematodes, even when one of the nicotinic receptor subtypes is dysfunctional due to the development of resistance. This is useful information for the formulation of commercial drugs that could prevent and overcome resistance.