Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA.
Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA, USA.
Int J Parasitol Drugs Drug Resist. 2020 Aug;13:38-50. doi: 10.1016/j.ijpddr.2020.04.002. Epub 2020 Apr 25.
Cholinergic agonists, like levamisole, are a major class of anthelmintic drugs that are known to act selectively on nicotinic acetylcholine receptors (nAChRs) on the somatic muscle and nerves of nematode parasites to produce their contraction and spastic paralysis. Previous studies have suggested that in addition to the nAChRs found on muscle and nerves, there are nAChRs on non-excitable tissues of nematode parasites. We looked for evidence of nAChRs expression in the cells of the intestine of the large pig nematode, Ascaris suum, using RT-PCR and RNAscope in situ hybridization and detected mRNA of nAChR subunits in the cells. These subunits include components of the putative levamisole receptor in A. suum muscle: Asu-unc-38, Asu-unc-29, Asu-unc-63 and Asu-acr-8. Relative expression of these mRNAs in A. suum intestine was quantified by qPCR. We also looked for and found expression of G protein-linked acetylcholine receptors (Asu-gar-1). We used Fluo-3 AM to detect intracellular calcium changes in response to receptor activation by acetylcholine (as a non-selective agonist) and levamisole (as an L-type nAChR agonist) to look for evidence of functioning nAChRs in the intestine. We found that both acetylcholine and levamisole elicited increases in intracellular calcium but their signal profiles in isolated intestinal tissues were different, suggesting activation of different receptor sets. The levamisole responses were blocked by mecamylamine, a nicotinic receptor antagonist in A. suum, indicating the activation of intestinal nAChRs rather than G protein-linked acetylcholine receptors (GARs) by levamisole. The detection of nAChRs in cells of the intestine, in addition to those on muscles and nerves, reveals another site of action of the cholinergic anthelmintics and a site that may contribute to the synergistic interactions of cholinergic anthelmintics with other anthelmintics that affect the intestine (Cry5B).
胆碱能激动剂，如左旋咪唑，是一大类驱虫药物，已知它们选择性地作用于线虫寄生虫的躯体肌肉和神经上的烟碱型乙酰胆碱受体 (nAChR)，导致其收缩和痉挛性瘫痪。先前的研究表明，除了在肌肉和神经上发现的 nAChR 之外，线虫寄生虫的非兴奋组织上也存在 nAChR。我们使用 RT-PCR 和 RNAscope 原位杂交技术在大猪蛔虫的肠道细胞中寻找 nAChR 表达的证据，并在细胞中检测到 nAChR 亚基的 mRNA。这些亚基包括在 A. suum 肌肉中的假定左旋咪唑受体的组成部分：Asu-unc-38、Asu-unc-29、Asu-unc-63 和 Asu-acr-8。通过 qPCR 定量检测这些 mRNA 在 A. suum 肠道中的相对表达量。我们还寻找并发现了 G 蛋白偶联乙酰胆碱受体 (Asu-gar-1) 的表达。我们使用 Fluo-3 AM 来检测乙酰胆碱（作为非选择性激动剂）和左旋咪唑（作为 L 型 nAChR 激动剂）激活受体后细胞内钙的变化，以寻找肠道中功能性 nAChR 的证据。我们发现乙酰胆碱和左旋咪唑都能引起细胞内钙的增加，但它们在分离的肠道组织中的信号特征不同，表明激活了不同的受体组。左旋咪唑的反应被美加明阻断，这是 A. suum 中的烟碱受体拮抗剂，表明左旋咪唑激活了肠道 nAChR，而不是 G 蛋白偶联乙酰胆碱受体 (GAR)。除了肌肉和神经上的 nAChR 之外，在肠道细胞中检测到 nAChR，揭示了另一个作用部位，即胆碱能驱虫剂的作用部位，并且可能有助于与影响肠道的其他驱虫剂（Cry5B）的协同相互作用。
