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

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 (BH4) is an essential cofactor for NOS activity. The present study tested the hypothesis that inhibition of endogenous BH4 rate-limiting enzyme GTP cyclohydrolase I (GTPCH I), and thus BH4 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, BH4 levels, GTPCH I and NOS mRNA, protein expression, and superoxide anion (O2*-) and ONOO- levels.

Results: Endogenous GTPCH I activity, BH4 levels, iNOS activity, and (O2*- and ONOO- levels were all augmented after ischemia/reperfusion. DAHP treatment significantly reduced GTPCH I activity, resulting in decreased BH4 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 microg/g tissue; n=5; P<0.05).

Conclusions: These results demonstrate that blockade of endogenous brain BH4 synthesis attenuates cerebral infarction via inhibiting iNOS and ONOO-, which may provide a mechanistic basis of novel therapeutic strategies for ischemic stroke.