Roszer Tamás, Józsa Tamás, Szentmiklósi A József, Bánfalvi Gáspár
Department of Animal Anatomy and Physiology, Faculty of Science, Debrecen University, Debrecen, Hungary.
Cell Tissue Res. 2009 May;336(2):325-35. doi: 10.1007/s00441-009-0764-3. Epub 2009 Mar 3.
Acetylcholine (ACh) is one of the main signals regulating nitric oxide synthase (NOS) expression and nitric oxide (NO) biosynthesis in mammals. However, few comparative studies have been performed on the role of ACh on NOS activity in non-mammalian animals. We have therefore studied the cholinergic control of NOS in the snail Helix pomatia and compared the effects of ACh on NO synthesis in the enteric nervous system of the snail and rat. Analyses by the NADPH-diaphorase reaction, immunocytochemistry, purification with ion-exchange chromatography, Western-blot, and quantitative polymerase chain reaction have revealed the expression of neuronal NOS in the rat intestine and of a 60-kDa subunit of NOS in the enteric nerve plexus of H. pomatia. In H. pomatia, quantification of the NO-derived nitrite ions has established that NO formation is confined to the NOS-containing midintestine. Nitrite production can be elevated by L-arginine but inhibited by N(omega)-nitro-L-arginine. In rats, ACh moderately elevates nitrite production, whereas ACh, the nicotinic receptor agonists (nicotine, acetyl thiocholine iodide, metacholine) and the cholinesterase inhibitor eserine reduce enteric nitrite formation in snails. The nicotinic receptor antagonist tubocurarine also provokes nitrite liberation, whereas the muscarinic receptor agonists or antagonists have no significant effect in snails. In the presence of EDTA or tetrodotoxin, ACh fails to inhibit nitrite production. In pharmacological studies, we have found that ACh contracts the midintestinal muscles and, in snails, simultaneously reduces the antagonistic muscle relaxant effect of L-arginine. Our experiments provide the first evidence for an inhibitory regulation of neuronal NO synthesis by ACh in an invertebrate species.
乙酰胆碱（ACh）是调节哺乳动物一氧化氮合酶（NOS）表达和一氧化氮（NO）生物合成的主要信号之一。然而，关于ACh在非哺乳动物中对NOS活性作用的比较研究较少。因此，我们研究了苹果螺（Helix pomatia）中NOS的胆碱能控制，并比较了ACh对蜗牛和大鼠肠神经系统中NO合成的影响。通过NADPH-黄递酶反应、免疫细胞化学、离子交换色谱纯化、蛋白质免疫印迹和定量聚合酶链反应分析发现，大鼠肠道中存在神经元型NOS的表达，而在苹果螺的肠神经丛中存在一种60 kDa的NOS亚基。在苹果螺中，对源自NO的亚硝酸盐离子的定量分析表明，NO的形成仅限于含有NOS的中肠。L-精氨酸可提高亚硝酸盐的产生，但N（ω）-硝基-L-精氨酸可抑制其产生。在大鼠中，ACh可适度提高亚硝酸盐的产生，而在蜗牛中，ACh、烟碱受体激动剂（尼古丁、碘化乙酰硫代胆碱、醋甲胆碱）和胆碱酯酶抑制剂毒扁豆碱可减少肠道亚硝酸盐的形成。烟碱受体拮抗剂筒箭毒碱也可引起亚硝酸盐释放，而毒蕈碱受体激动剂或拮抗剂对蜗牛没有显著影响。在存在乙二胺四乙酸（EDTA）或河豚毒素的情况下，ACh无法抑制亚硝酸盐的产生。在药理学研究中，我们发现ACh可使中肠肌肉收缩，并且在蜗牛中，同时降低L-精氨酸的拮抗肌肉松弛作用。我们的实验首次证明了在无脊椎动物中ACh对神经元型NO合成具有抑制调节作用。
