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 remains 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 protective 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 aziridinium both in vivo and in vitro. For the in vivo evaluation rats received bilateral intracerebroventricular injections of ethylcholine aziridinium (2nmol/ventricle) or vehicle. In the hippocampus a transient decrease in nitric oxide synthase activity occurred, reaching its lowest levels three days after ethylcholine aziridinium treatment (51.7+/-9.8% of controls). The decrease coincided with the maximal reduction in choline acetyltransferase activity as marker for the extent of cholinergic lesion. The effect of pharmacological inhibition of nitric oxide synthase was tested by application of various nitric oxide synthase inhibitors with different selectivity for the nitric oxide synthase-isoforms. Unspecific nitric oxide synthase inhibition resulted in a significant potentiation of the loss of choline acetyltransferase activity in the hippocampus measured seven days after ethylcholine aziridinium application, whereas the specific inhibition of neuronal or inducible nitric oxide synthase was ineffective. These pharmacological data are suggestive for a neuroprotective role of nitric oxide generated by endothelial nitric oxide synthase. In vitro experiments were performed using serum-free primary neuronal cell cultures from hippocampus, cortex and septum of E15-17 Wistar rat embryos. Ethylcholine aziridinium-application in a range of 5-80microM resulted in delayed apoptotic neurodegeneration with a maximum after three days as confirmed by morphological criteria, life-death assays and DNA laddering. Nitric oxide synthase activity in harvested cells decreased in a dose- and time-dependent manner. Nitric oxide production as determined by measurement of the accumulated metabolite nitrite in the medium was equally low in controls and in ethylcholine aziridinium treated cells (range 0.77-1.86microM nitrite). An expression of inducible nitric oxide synthase messenger RNA could not be detected by semiquantitative RT-PCR 13h after ethylcholine aziridinium application. The present data indicate that in a model of delayed apoptotic neurodegeneration as induced by ethylcholine aziridinium neuronal cell death in vitro and in vivo is independent of the cytotoxic potential of nitric oxide. This is confirmed by a decrease in nitric oxide synthase activity, absence of nitric oxide production and absence of inducible nitric oxide synthase expression. 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 methylester.
Lautenschlager
M
M
Institute of Pharmacology and Toxicology, Medical Faculty Charité, Humboldt-University Berlin, Dorotheenstrasse 94, D-10098, Berlin, Germany.
Onufriev
M V
MV
Gulyaeva
N V
NV
Harms
C
C
Freyer
D
D
Sehmsdorf
U
U
Ruscher
K
K
Moiseeva
Y V
YV
Arnswald
A
A
Victorov
I
I
Dirnagl
U
U
Weber
J R
JR
Hörtnagl
H
H
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001388:Aziridines / Q000008:administration & dosage / Q000633:toxicity*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D002552:Cerebral Ventricles / Q000187:drug effects / Q000502:physiology; D002794:Choline / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000633:toxicity; D002795:Choline O-Acetyltransferase / Q000378:metabolism; D005333:Fetus; D005625:Frontal Lobe / Q000187:drug effects / Q000502:physiology* / Q000503:physiopathology; D006624:Hippocampus / Q000187:drug effects / Q000502:physiology* / Q000503:physiopathology; D007276:Injections, Intraventricular; D008297:Male; D009410:Nerve Degeneration / Q000139:chemically induced / Q000473:pathology / Q000503:physiopathology*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D051381:Rats; D017207:Rats, Sprague-Dawley; D020665:Septum of Brain / Q000166:cytology; D014118:Toxins, Biological / Q000633:toxicity
D001388:Aziridines; D014118:Toxins, Biological; D009569:Nitric Oxide; C044894:ethylcholine aziridinium; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D002795:Choline O-Acetyltransferase; D002794:Choline