β-alanine betaine and nAChRs in Ascaris

Abstract

Anthelmintic drugs are used to control soil-transmitted helminths that infect a third of the worlds human population. There is increasing concern about the development of resistance to anthelmintic drugs because of the limited number of compounds available and there is an unmet need for new resistance-busting drugs. Here we describe the presence of a previously unrecognized endogenous acetylcholine analogue, {beta}-alanine betaine, which may serve as an endogenous ligand for an alternate subfamily of nicotinic receptors (DEG-3/DES-2) that could be developed as novel drug targets because their analogues are not present in their human or animal hosts. We collected peri-enteric fluid from female Ascaris suum (a model for the human parasite, Ascaris lumbricoides) and subjected it to chromatography and MS/MS to reveal signals consistent with acetylcholine, choline, and {beta}-alanine betaine but we did not recover betaine. We injected betaine into female Ascaris suum which produced no effect. However, injection of {beta}-alanine betaine, produced characteristic pretzel coiling and injection of levamisole produced a rod-like spastic paralysis. The differences between {beta}-alanine betaine and levamisole suggested that they activate different nAChRs subfamilies. PCR showed that messages of the DEG-3 subfamily of nAChR channels, which are betaine targets and were present in the intestine and body wall of A. suum. Calcium signaling experiments showed that {beta}-alanine betaine increased intracellular calcium of the intestine enterocytes and electrophysiology of the body muscle cells demonstrated that {beta}-alanine betaine produced membrane potential depolarization. In N2 elegans, application of {beta}-alanine betaine produced gradual inhibition of motility, which was reduced in acr-20, acr-23, des-2, deg-3 and lgc-41 null-mutants. These observations suggest that, in addition to acetylcholine, {beta}-alanine betaine - an anaerobic analog of betaine - may function as an endogenous ligand in anaerobic nematodes such as A. suum. An expanded repertoire of nicotinic acetylcholine receptor subfamilies in nematodes relative to mammals may reflect a corresponding need for diversification of cholinergic endogenous ligands in these organisms. This repertoire could allow their simpler neuronal system to perform more complex controls and be exploited for development of different and novel subfamily selective cholinergic anthelmintics. Author SummaryThere is increasing concern about the development of resistance to anthelmintic drugs because of the limited number of compounds available and there is an unmet need for new resistance-busting drugs. The cholinergic anthelmintics are one of the three major classes of anti-nematodal drugs that are used for control and treatment of soil-transmitted helminths. Each of these cholinergic anthelmintics (levamisole, pyrantel, derquantel, monepantel and oxantel) are selective for different nematode nicotinic acetylcholine receptors (nAChRs). The differences in selectivity could explain why resistance and species sensitivities varies across the different cholinergic anthelmintics. It is surprising how many nAChR genes are expressed in nematodes with more being present compared to humans. Why is this? Could it be that there are also more endogenous ligands other than acetylcholine allowing their simpler neuronal system to perform more complex control? We looked for additional analogues of acetylcholine in the body fluid of the large intestinal parasite of the pig Ascaris suum (a model for Ascaris lumbricoides) and identified the anaerobic cholinergic compound {beta}-alanine betaine. We found evidence that suggests that {beta}-alanine betaine may serve as an endogenous ligand for an alternate subfamily of nicotinic receptors (DEG-3/DES-2) that could be developed as novel drug targets because their receptor analogues are not present in human or animal hosts.