A novel anaerobic cholinergic signaling compound, β-alanine betaine, and nAChRs in Ascaris.

Anthelmintic drugs are used to control soil-transmitted helminths that infect a quarter of the world's 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 (ACh) analogue, β-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 pseudocoelomic fluid from female Ascaris suum (a model for the human parasite Ascaris lumbricoides) and subjected it to HILIC-MS and HILIC-MS/MS to reveal signals consistent with ACh, choline, and β-alanine betaine but not betaine, propionylcholine, butyrylcholine, or butyrobetaine. The presence of β-alanine betaine was confirmed by comparison with an analytical standard. Injection of betaine into female A. suum produced no effect. However, injection of β-alanine betaine produced characteristic pretzel coiling and injection of levamisole produced a rod-like spastic paralysis. The differences between β-alanine betaine and levamisole suggested that they activate different nAChRs subfamilies. RT-PCR showed that message for members of the DEG-3/DES-2 subfamily of nAChR channels, which are betaine targets, were present in the intestine and body wall of A. suum. β-alanine betaine increased intracellular Ca2+ of the intestinal enterocytes and depolarized the membrane potential of body wall muscle cells. In N2 Caenorhabditis elegans, application of β-alanine betaine produced gradual inhibition of motility, which was reduced in acr-20, acr-23, deg-3/des-2 and lgc-41 null-mutants. These observations suggest that, in addition to ACh, β-alanine betaine - an analog of betaine produced under anaerobic conditions - 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.