[H-3]L-N-G-nitroarginine binding after transient focal ischemia and NMDA-induced excitotoxicity in type I and type III nitric oxide synthase null mice

We investigated the density and distribution of nitric oxide synthase (NOS) binding by quantitative autoradiography using [H-3]L-N-G-nitroarginine ([H-3]L-NNA) after transient focal ischemia or intrastriatal injection of N-methyl-D-aspartate (NMDA) in wild-type (SV-129 and C57black/6) and type I (neuronal) and type III (endothelial) NOS-deficient mice. The middle cerebral artery (MCA) was occluded by an intraluminal filament for 3 h followed by 10 min to 7 days of reperfusion. Specific [H-3]L-NNA binding, observed in the wild-type and type III mutant mouse at baseline, increased by 50-250% in the MCA territory during ischemia and the first 3 h of reperfusion. The density of binding sites (B-max), but not the dissociation constant (K-d), increased significantly during the ischemic period as did type I NOS mRNA as detected by quantitative reverse transcription polymerase chain reaction. [H-3]L-NNA binding after intrastriatal NMDA injection also increased by 20-230%. In the type I NOS-deficient mouse, [H-3]L-NNA binding was low and only a very small increase was observed after ischemia or excitotoxicity. Under conditions of this study, [3H]L-NNA did not bind to type II NOS as there was no difference in the distribution or density of [H-3]L-NNA binding in the rat spleen obtained after lipopolysaccharide treatment despite induction of NOS type II catalytic activity. Our data suggest that an ischemic/excitotoxic insult up-regulates type I NOS gene expression and [H-3]L-NNA binding and that this up-regulation may play a pivotal role in the pathogenesis of ischemic/excitotoxic diseases.