Provider: - Institution: - Data provided by Europeana Collections- GAergic and cholinergic systems in the neuromuscular system of the parasitic nematodes are the main target of action for anthelmintic drugs. In the nematode cholinergic system pharmacological importance has primarily nicotinic-acetylcholine receptor (nAChR). On the other hand, the GABA-receptor was originally identified as the main site of action for the Avermectins and Milbemicins. However, later was shown that these drugs work on a new, previously never described glutamate-gated chloride ion channel, located in the nematode pharynx. The most important problems that are endangering the success of antiparasitic therapy are the development of resistance in the parasites and often occurrences of the toxic effects of antiparasitic drugs in host. In the order to elucidate the mechanisms of action of GABAergičkih and cholinergic anthelmintic, we examined the pharmacological characteristics of the representatives of these two groups of drugs in the isolated nerve-muscle preparation of large pig nematode Ascaris suum. Also, it was important to examine comparatively differences in the effects of cholinergic and GABAergic anthelmintic on the corresponding receptors in mammals (investigations were performed on isolated rat diaphragm and ileum), and thus to analyze the mechanisms of their adverse effects. In presented investigations we measured the effects of contraction or relaxation on isolated nematode or mammalian preparations and analyzed the results by using appropriate statistical methods (non-linear regression, ANOVA, t-test). Based on the results obtained in our investigations the following conclusions may be presented: (1) Agonists of L, N and B type of nematode nicotinic acetylcholine receptor (nAChR) studied on a model of neuromuscular preparations of A. suum, showed variable efficacy. Highest efficiency in the first group of tested agonists has pyrantel (agonist of L-type nAChR, EC50=0.010 μM, Emax=2.5g), then bephenium (agonist of B-type nAChR, EC50=0.37μM, Emax=2.7g) and at the end acetylcholine (an endogenous neurotransmitter, agonists of all three types of receptors L, N and B, EC50=6.12-6.45μM, Emax=1.71-2.07g). Highest efficiency in the second group of nicotinic receptor agonists has been demonstrated by tribendimidine (probably agonist of L-type nAChR, EC50=0.064μM, Emax=1.29g), followed by levamisole (agonist of L-type nAChR, EC50=0.34μM, Emax=0.68g) and at the end nicotine (an agonist of N-type nAChR, EC50=4.99μM, Emax=1.07g). (2) Nicotinic acetylcholine receptor of A. suum, that acting tribendimidine and nicotine, exhibit characteristics of both types of mammalian nAChRs: a) the characteristics of mammalian muscle type of nAChR, because it is sensitive primarily to pancuronium but also to tubocurarine, and b) the characteristics of neuronal nAChRs of mammals, because it is sensitive primarily to mecamylamine but also to hexamethonium. (3) GABA induced a dose-dependent relaxation of A. suum neuromuscular preparation, the value of the mean EC50 was 7.40μM, while piperazine causes relaxation with EC50 of 331μM. Receptor through which GABA and piperazine exhibit relaxation of A. suum is different from mammalian GABAA receptor, because it is insensitive to bicuculine (specific mammalian GABAA receptor antagonist). (4) Ivermectin and moxidectin by themselves do not lead to the relaxation of neuromuscular preparation of A. suum. Furthermore, ivermectin does not potentiate the relaxation caused by GABA. Ivermectin induces relaxation only when is administered after GABA or in the neuromuscular preparations of A. suum with high amplitude of spontaneous activity (spontaneous contraction and relaxation about 0.5g). Therefore, ivermectin for its action requires prior activation of GABA-dependent chloride channels. (5) Carvacrol 100 and 300μM, causes long-term relaxation of neuromuscular preparation of A. suum. Carvacrol significantly potentiates relaxation caused by GABA and significantly inhibits the dose-dependent contractile effects of ACh, shifting the EC50 value from control 5.22μM to the 13.88μM and 22.72μM, respectively. Carvacrol reduced the maximum contractile effect of ACh from control 3.1g to the 2.50g and 1.96 g, respectively. Therefore, this is probably a classical non-competitive physiological antagonism, which carvacrol manifests by itself in relation to ACh. (6) Neither one combination of different parameters of Electrical Field Stimulation (EFS) (10Hz to 100Hz, 0.01-1.0ms, 5 to 30V, from 2.0 sec. to continuous stimulation), do not causes indirect stimulation of the A. suum neuromuscular preparation (contractions are insensitive to mecamylamine). The explanation may be in greater sensitivity of muscle contractile machinery on EFS, compared to the sensitivity of nerves that release acetylcholine. Contractions obtained from EFS were still sensitive to 10 and 30μM GABA, which can be explained by strong hyperpolarization of muscle and nerve cells that GABA causes. (7) GABA at concentration of 1μM increases twice the EC50 value of ACh from 4.42μM to 10.94μM, with no significant changes in Emax. Ivermectin 300nM does not change significantly the value of EC50 of ACh (10.26μM compared to the control 8.09μM), but decreases the value of Emax for 0.5g. When applied together, ivermectin and GABA increased EC50 value of ACh from 8.28 to 50.13μM and reduced the value of Emax from 2.51 to 1.90g. The combination of GABA and ivermectin exerts a highly significant inhibitory effect on ACh-induced contractions at all concentrations in which it is applied, whereby the effect of the combination is greater than their individual effects. (8) Piperazine at concentration of 100μM does not change the EC50 value of ACh (10.76μM compared to control 9.47μM), but decreases the value of Emax for almost 1g (from 4.38g to 3.45g). When ivermectin (300nM) applied with piperazine, EC50 value of ACh still does not change, but the Emax decreased to 2.73g. Reduction of the Emax without changing the EC50 exhibit non-competitive antagonists, as piperazine and ivermectin act in relation to the effect of ACh. (9) Nematode nicotinic receptor agonists - levamisole, pyrantel and bephenium act antagonistically to the contractions of isolated rat diaphragm induced by the EFS. IC50 of levamisole was 53.83μM, bephenium 54.47μM and pyrantel 5.53μM. All three tested drugs are expressed presynaptic effect as well, levamisole at concentrations above 30μM, bephenium at concentrations higher than 10μM and pyrantel at concentrations above 5μM. Tribendimidine did not show any effect on the isolated diaphragm contractions at concentrations up to 300μM, neither presynaptic nor postsynaptic. (10) GABA receptor agonists - piperazine (30 to 3000μM) and moxidectin (10 to 30μM) did not show any effect on the isolated diaphragm contractions induced by EFS. (11) Nematode nicotinic receptor agonists - tribendimidine, bephenium, levamisole and pyrantel (1 to 100μM) did not show any effect on the basal tonus of isolated rat ileum. Applied at concentrations of 10 and 100μM tested drugs as well did not change the mean contraction of the ileum induced by the EFC. GABA-receptor agonists, piperazine (30 to 1000μM) and moxidectin (1 to 100μM) did not show any effects on the basal tonus of isolated rat ileum, or changes the mean contractions of the ileum induced by the EFC. (12) Carvacrol (10, 30 and 100μM) does not change the mean contractions of rat diaphragm induced by the EFC. However, the highest tested concentration leads to some level of tetanic fade, suggesting a potential presynaptic effect of carvacrol. On isolated rat ileum, carvacrol (300 to 1000μM) does not affect the contractions caused with EFS. The presented findings suggest that some nematode nAChR agonists exhibit a different efficiency on neuromuscular preparation of A. suum and that the efficiency obviously depends on the subtype of receptor that drug predominantly acts on (L, B or N). The combination of drugs, agonists of different subtypes of nAChR could represent a completely new approach to the treatment of parasitic infections, compared to conventional therapy with a single drug. Tribendimidin, according to the results obtained is very potent and potentially useful antiparasitic drug in veterinary medicine. At the same time it does not work on peripheral mammalian cholinergic and GABA-ergic nerve structure, which indicates high safety of its application in the host. According our results GABA-ergic macrolide anthelmintics, by themselves, do not cause muscle relaxation in A. suum, but after action of GABA they exhibit relaxant effect. Obviously, this effect required previous activation of chloride channels, which open GABA-gating. Piperazine on the other hand, by itself causes relaxation, but most likely not with action on the same type of receptor as Avermectins and Milbemicins. However, along with them, piperazine express noncompetitive antagonism to the contractile effects of ACh, therefore this may be a new combination of drugs in the pharmacotherapy of parasitic infections. Based on the effects of carvacrol in our research and chemical characteristics of this compound of natural origin, we can conclude that carvacrol is a serious candidate for a potential antihelmintic drug. It could be applied alone, or in combination with GABA agonists whose antiparasitic effect increases (as we showed in our investigations). Finally, all tested drugs are exhibited little or no toxic potential in the models of rat isolated diaphragm and ileum. If they do not pass the blood-brain barrier (which is evidenced for all of them under physiological conditions), and therefore does not affect the central GABA and cholinergic structures, their toxicity for host is very low, because peripheral nicotinic and GABA structures they affect only in extremely high concentrations (which far exceed therapeutic).- Gabaergički i holinergički sistem u neuro-miši