Role of nitric oxide in the ethylcholine aziridinium model of delayed apoptotic neurodegeneration in vivo and in vitro

The involvement of nitric oxide in neurodegenerative processes still remain s incompletely characterized. Although nitric oxide has been reported to be an important mediator in neuronal degeneration in different models of cell death involving NMDA-receptor activation, increasing evidence for protecti ve mechanisms has been obtained. In this study the role of nitric oxide was investigated in a model of NMDA-independent, delayed apoptotic cell death, induced by the neurotoxin ethylcholine aziridinium ethylcholine aziridiniu m both in vivo and in vitro. For the in vivo evaluation rats received bilat eral intracerebroventricular injections of ethylcholine aziridinium (2nmol/ ventricle) or vehicle. In the hippocampus a transient decrease in nitric ox ide synthase activity occurred, reaching its lowest levels three days after ethylcholine aziridinium treatment (51.7 +/- 9.8% of controls). The decrea se coincided with the maximal reduction in choline acetyltransferase activi ty as marker for the extent of cholinergic lesion. The effect of pharmacolo gical inhibition of nitric oxide synthase was tested by application of vari ous nitric oxide synthase inhibitors with different selectivity for the nit ric oxide synthase-isoforms. Unspecific nitric oxide synthase inhibition re sulted in a significant potentiation of the loss of choline acetyltransfera se activity in the hippocampus measured seven days after ethylcholine aziri dinium application, whereas the specific inhibition of neuronal or inducibl e nitric oxide synthase was ineffective. These pharmacological data are sug gestive for a neuroprotective role of nitric oxide generated by endothelial nitric oxide synthase. In vitro experiments were performed using serum-fre e primary neuronal cell cultures from hippocampus, cortex and septum of E15 -17 Wistar rat embryos. Ethylcholine aziridinium-applicatian in a range of 5-80 mu M resulted in delayed apoptotic neurodegeneration with a maximum af ter three days as confirmed by morphological criteria, life-death assays an d DNA laddering. Nitric oxide synthase activity in harvested cells decrease d in a dose- and time-dependent manner. Nitric oxide production as determin ed by measurement of the accumulated metabolite nitrite in the medium was e qually low in controls and in ethylcholine aziridinium treated cells (range 0.77-1.86 mu M nitrite). An expression of inducible nitric oxide synthase messenger RNA could not be detected by semiquantitative RT-PCR 13 h after e thylcholine aziridinium application. The present data indicate that in a model of delayed apoptotic neurodegener ation as induced by ethylcholine aziridinium neuronal cell death in vitro a nd in vivo is independent of the cytotoxic potential of nitric oxide. This is confirmed by a decrease in nitric oxide synthase activity, absence of ni tric oxide production and absence of inducible nitric oxide synthase expres sion. In contrast, evidence for a neuroprotective role of nitric oxide was obtained in vivo as indicated by the exaggeration of the cholinergic lesion after unspecific nitric oxide synthase inhibition by N-nitro-L-arginine me thylester. (C) 2000 IBRO. Published by Elsevier Science Ltd.