Stoichiometric Relationships Between Endothelial Tetrahydrobiopterin, Endothelial NO Synthase (eNOS) Activity, and eNOS Coupling in Vivo

Endothelial dysfunction in vascular disease states is associated with reduced NO bioactivity and increased superoxide (O 2 ·− ) production. Some data suggest that an important mechanism underlying endothelial dysfunction is endothelial NO synthase (eNOS) uncoupling, whereby eNOS generates O 2 ·− rather than NO, possibly because of a mismatch between eNOS protein and its cofactor tetrahydrobiopterin (BH4). However, the mechanistic relationship between BH4 availability and eNOS coupling in vivo remains undefined because no studies have investigated the regulation of eNOS by BH4 in the absence of vascular disease states that cause pathological oxidative stress through multiple mechanisms. We investigated the stoichiometry of BH4–eNOS interactions in vivo by crossing endothelial-targeted eNOS transgenic (eNOS-Tg) mice with mice overexpressing endothelial GTP cyclohydrolase 1 (GCH-Tg), the rate-limiting enzyme in BH4 synthesis. eNOS protein was increased 8-fold in eNOS-Tg and eNOS/GCH-Tg mice compared with wild type. The ratio of eNOS dimer:monomer was significantly reduced in aortas from eNOS-Tg mice compared with wild-type mice but restored to normal in eNOS/GCH-Tg mice. NO synthesis was elevated by 2-fold in GCH-Tg and eNOS-Tg mice but by 4-fold in eNOS/GCH-Tg mice compared with wild type. Aortic BH4 levels were elevated in GCH-Tg and maintained in eNOS/GCH-Tg mice but depleted in eNOS-Tg mice compared with wild type. Aortic and cardiac O 2 ·− production was significantly increased in eNOS-Tg mice compared with wild type but was normalized after NOS inhibition with N ω-nitro- l -arginine methyl ester hydrochloride (L-NAME), suggesting O 2 ·− production by uncoupled eNOS. In contrast, in eNOS/GCH-Tg mice, O 2 ·− production was similar to wild type, and L-NAME had no effect, indicating preserved eNOS coupling. These data indicate that eNOS coupling is directly related to eNOS–BH4 stoichiometry even in the absence of a vascular disease state. Endothelial BH4 availability is a pivotal regulator of eNOS activity and enzymatic coupling in vivo.