We propose that neuronal production of nitric oxide (-NO) provides a common link between two seemingly independent mechanisms of brain injury -- excitatory neurotransmitters and oxygen radicals. The connection results from the recent demonstration that glutamate stimulates neurons to produce -NO. Neuronal -NO helps regulate local cerebral blood flow (CBF) as well as plays an essential role in synaptic plasticity and the normal development of brain. We hypothesize that ischemia initiates events that mimic the normal physiological regulation of the N-methyl-D-aspartate subclass of glutamate receptors, causing overproduction of -NO when tissue is reoxygenated. Oxidative metabolism disrupted by ischemia will produce oxygen radicals that react with the elevated concentrations of -NO, thereby greatly accelerating the formation of the destructive and strongly oxidizing species peroxynitrite (ONOO-) and intensifying cerebral injury. Nitric oxide is produced by the oxidation of arginine, and we and others have shown that brain -NO production is rapidly inhibited in vivo by the substrate analog, nitroarginine. Nitroarginine decreases CBF in control rats by 40% without a direct anesthetic action. Nitroarginine also attenuates CBF increases caused by bicuculline-induced seizures and reduces cortical infarct volume when administered at the end of the ischemic period. In the previous supported period, we found that 10,000 U/kg polyethylene-glycol conjugated superoxide dismutase (PEG-SOD) reduced infarct volume in a rat middle cerebral artery (MCA) stroke model. Recently, Dr. Hsu (collaborator) found that the protection was lost at a three fold higher dosage of PEG-SOD. Similar results are observed in myocardial ischemia, even though PEG-SOD is not directly toxic to either organ. We have shown that PEG-SOD catalyzes the nitration of protein tyrosines by peroxynitrite, and propose that this toxic side-reaction may account for the loss of efficacy at high PEG-SOD dosages.
The Specific Aims of this proposal are to measure -NO, N02 & ONOO- production resulting from ischemia/reperfusion of brain, examine whether low dosages of nitroarginine will reduce cortical infarct volume in a rat MCA-carotid occlusion stroke model, and determine the potential role of peroxynitrite formation in the paradoxical loss of SOD efficacy at high dosages. We have developed a novel SOD which does not nitrate phenolics in the presence of peroxynitrite and will test whether it is more efficacious. We have also raised antibodies to identify proteins nitrated by SOD plus peroxynitrite in ischemic brain, which will help delineate regions producing reactive nitrogen species. The hypothesis is consistent with current evidence concerning cerebral injury, makes testable predictions and offers new therapeutic approaches.
