Zandecki M, Raeymaekers P, Janssens J, Tack J, Vanden Berghe P
Centre for Gastroenterological Research, Katholieke Universiteit Leuven, Belgium.
Neurogastroenterol Motil. 2006 Apr;18(4):307-15. doi: 10.1111/j.1365-2982.2005.00755.x.
Previously, we demonstrated that intestinal inflammation leads to a postinflammatory loss of nitric oxide synthase (NOS)-expressing myenteric neurones and motility disturbances. Here, we investigated whether high NO concentrations could be responsible for the decrease in NOS neurones. Myenteric neurone cultures, prepared from guinea-pig small intestine, were incubated with NO donors [sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1)]. After fixation, NOS neurones were identified by NADPH diaphorase staining and neurone-specific enolase (NSE)-positive neuronal content was assessed with an enzyme-linked immunosorbent assay (ELISA)-based method. Twenty-four hours incubation with SIN-1 (10(-3) mol L(-1)) or SNP (10(-4) mol L(-1) or higher) reduced the number of NADPH diaphorase-positive neurones. SNP incubation did not affect the NSE-positive neuronal content. Shorter incubations (SNP: 4 and 12 h) had no significant effect. The SNP-induced reduction was reversed by glutathione (GSH), but not by NO- or O-scavengers, whereas GSH depletion enhanced the decrease. The NO-dependent guanylate cyclase-blocker 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) did not affect the SNP effect. This reduction can be explained by either specific apoptosis of NOS neurones or downregulation of NOS activity. However, TdT-mediated X-dUTP nick end labelling (TUNEL stainings argue in favour of the latter. In conclusion, the NO donor SNP decreases the number of NOS-expressing myenteric neurones time and concentration dependently, without affecting the amount of neuronal material. Glutathione plays an important protective role.
先前，我们证明肠道炎症会导致炎症后一氧化氮合酶（NOS）表达的肌间神经元丧失以及运动障碍。在此，我们研究了高浓度一氧化氮（NO）是否可能是导致NOS神经元减少的原因。从豚鼠小肠制备的肌间神经元培养物与NO供体[硝普钠（SNP）和3-吗啉代亚硝基胍（SIN-1）]一起孵育。固定后，通过NADPH黄递酶染色鉴定NOS神经元，并用基于酶联免疫吸附测定（ELISA）的方法评估神经元特异性烯醇化酶（NSE）阳性神经元含量。用SIN-1（10⁻³ mol/L）或SNP（10⁻⁴ mol/L或更高）孵育24小时可减少NADPH黄递酶阳性神经元的数量。SNP孵育不影响NSE阳性神经元含量。较短时间的孵育（SNP：4小时和12小时）没有显著影响。SNP诱导的减少可被谷胱甘肽（GSH）逆转，但不能被NO或O清除剂逆转，而GSH耗竭会增强这种减少。NO依赖性鸟苷酸环化酶阻滞剂1H-[1,2,4]恶二唑并[4,3-a]喹喔啉-1-酮（ODQ）不影响SNP的作用。这种减少可以通过NOS神经元的特异性凋亡或NOS活性的下调来解释。然而，末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）染色支持后者。总之，NO供体SNP可时间和浓度依赖性地减少表达NOS的肌间神经元数量，而不影响神经元物质的量。谷胱甘肽起着重要的保护作用。
