Endothelial nitric oxide synthase dysfunction in diabetic mice: importance of tetrahydrobiopterin in eNOS dimerisation.
OBJECTIVE
Impaired nitric oxide (NO) bioactivity and increased superoxide (SO) production are characteristics of vascular endothelial dysfunction in diabetes. The underlying mechanisms remain unknown. In this regard, we investigated the role of tetrahydrobiopterin (BH4) bioavailability in regulating endothelial nitric oxide synthase (eNOS) activity, dimerisation and SO production in streptozotocin-induced diabetic mice.


METHODS
Mouse aortas were used for assays of the following: (1) aortic function by isometric tension; (2) NO by electronic paramagnetic resonance; (3) SO by lucigenin-enhanced chemiluminescence and dihydroethidine fluorescence; (4) total biopterin and BH4 by high-performance liquid chromatography; and (5) eNOS protein expression and dimerisation by immunoblotting.


RESULTS
In diabetic mouse aortas, relaxations to acetylcholine and NO levels were significantly decreased, but SO production was increased, in association with reductions in total biopterins and BH4. Although total eNOS levels were increased in diabetes, the protein mainly existed in monomeric form. Conversely, specifically augmented BH4 in diabetic endothelium preserved eNOS dimerisation, but the expression remained unchanged.


CONCLUSIONS
Our results demonstrate that BH4 plays an important role in regulating eNOS activity and its functional protein structure, suggesting that increasing endothelial BH4 and/or protecting it from oxidation may be a rational therapeutic strategy to restore eNOS function in diabetes.
Cai
S
S
Department of Cardiovascular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DU, UK.
Khoo
J
J
Mussa
S
S
Alp
N J
NJ
Channon
K M
KM
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology*; D019281:Dimerization; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009599:Nitroprusside / Q000494:pharmacology; D010656:Phenylephrine / Q000494:pharmacology; D012016:Reference Values; D013481:Superoxides / Q000378:metabolism
D013481:Superoxides; D009599:Nitroprusside; D010656:Phenylephrine; D001708:Biopterins; C003402:sapropterin; D000109:Acetylcholine