The effect of nitric oxide donors on nitric oxide synthase-expressing myenteric neurones in culture.
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.
Centre for Gastroenterological Research, Katholieke Universiteit Leuven, Belgium.
M
M
Zandecki
P
P
Raeymaekers
J
J
Janssens
J
J
Tack
P
P
Vanden Berghe
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D016166:Free Radical Scavengers / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D006168:Guinea Pigs; D020287:In Situ Nick-End Labeling; D066298:In Vitro Techniques; D009197:Myenteric Plexus / Q000187:drug effects / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000378:metabolism
D016166:Free Radical Scavengers; D020030:Nitric Oxide Donors; D019001:Nitric Oxide Synthase; D009252:NADPH Dehydrogenase; D010751:Phosphopyruvate Hydratase; D005978:Glutathione