Inhibition of Brain GTP Cyclohydrolase I and Tetrahydrobiopterin Attenuates Cerebral Infarction via Reducing Inducible NO Synthase and Peroxynitrite in Ischemic Stroke

Abstract

Background— Inducible NO synthase (NOS)–derived peroxynitrite (ONOO − ) during ischemia/reperfusion contributes to ischemic brain injury. However, inducible NOS (iNOS) regulation in ischemic stroke remains unknown. Tetrahydrobiopterin (BH 4 ) is an essential cofactor for NOS activity. The present study tested the hypothesis that inhibition of endogenous BH 4 rate-limiting enzyme GTP cyclohydrolase I (GTPCH I), and thus BH 4 synthesis, reduces cerebral infarction via inhibiting iNOS and ONOO − in transient focal ischemia. Methods— Focal ischemia (2 hours) was created in adult male Sprague-Dawley rats (250 to 300 g) by middle cerebral artery occlusion (MCAO). Rats were treated 12 hours before MCAO with vehicle or diamino-6-hydroxypyrimidine (DAHP; 0.5 g/kg IP), a selective GTPCH I inhibitor. Brains were harvested 24 hours after reperfusion for assays of infarct volume, blood–brain barrier (BBB) permeability, GTPCH I activity, BH 4 levels, GTPCH I and NOS mRNA, protein expression, and superoxide anion (O 2 · − ) and ONOO − levels. Results— Endogenous GTPCH I activity, BH 4 levels, iNOS activity, and (O 2 · − and ONOO − levels were all augmented after ischemia/reperfusion. DAHP treatment significantly reduced GTPCH I activity, resulting in decreased BH 4 levels, iNOS activity, and ONOO − levels. Consequently, DAHP treatment significantly reduced the infarct size compared with the nontreated group (22.3±5.6 versus 38.3±7.4%; n=6; P <0.05). Similarly, BBB permeability was significantly reduced after DAHP pretreatment compared with the control group (4.11±0.22 versus 7.78±0.44 μg/g tissue; n=5; P <0.05). Conclusion— These results demonstrate that blockade of endogenous brain BH 4 synthesis attenuates cerebral infarction via inhibiting iNOS and ONOO − , which may provide a mechanistic basis of novel therapeutic strategies for ischemic stroke.