Hippocampal neurons in organotypic slice culture are highly resistant to damage by endogenous and exogenous nitric oxide.
Nitric oxide (NO) has been proposed to mediate neurodegeneration arising from NMDA receptor activity, but the issue remains controversial. The hypothesis was re-examined using organotypic slice cultures of rat hippocampus, with steps being taken to avoid known artefacts. The NO-cGMP signalling pathway was well preserved in such cultures. Brief exposure to NMDA resulted in a concentration-dependent delayed neuronal death that could be nullified by administration of the NMDA antagonist MK801 (10 microm) given postexposure. Two inhibitors of NO synthesis failed to protect the slices, despite fully blocking NMDA-induced cGMP accumulation. By comparing NMDA-induced cGMP accumulation with that produced by an NO donor, toxic NMDA concentrations were estimated to produce only physiological NO concentrations (2 nm). In studies of the vulnerability of the slices to exogenous NO, it was found that continuous exposure to up to 4.5 microm NO failed to affect ATP levels (measured after 6 h) or cause damage during 24 h, whereas treatment with the respiratory inhibitors myxothiazol or cyanide caused ATP depletion and complete cell death within 24 h. An NO concentration of 10 microm was required for ATP depletion and cell death, presumably through respiratory inhibition. It is concluded that sustained activity of neuronal NO synthase in intact hippocampal tissue can generate only low nanomolar NO concentrations, which are unlikely to be toxic. At the same time, the tissue is remarkably resistant to exogenous NO at up to 1000-fold higher concentrations. Together, the results seriously question the proposed role of NO in NMDA receptor-mediated excitotoxicity.
Keynes
Robert G
RG
Wolfson Institute for Biomedical Research, University College London, Cruciform Building, Gower Street, London WC1E 6BT, UK.
Duport
Sophie
S
Garthwaite
John
J
D000818:Animals; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D006152:Cyclic GMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006624:Hippocampus / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology*; D009924:Organ Culture Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley
D009569:Nitric Oxide; D016202:N-Methylaspartate; D006152:Cyclic GMP