Ratio of 5,6,7,8-tetrahydrobiopterin to 7,8-dihydrobiopterin in endothelial cells determines glucose-elicited changes in NO vs. superoxide production by eNOS.
5,6,7,8-Tetrahydrobiopterin (BH(4)) is an essential cofactor of nitric oxide synthases (NOSs). Oxidation of BH(4), in the setting of diabetes and other chronic vasoinflammatory conditions, can cause cofactor insufficiency and uncoupling of endothelial NOS (eNOS), manifest by a switch from nitric oxide (NO) to superoxide production. Here we tested the hypothesis that eNOS uncoupling is not simply a consequence of BH(4) insufficiency, but rather results from a diminished ratio of BH(4) vs. its catalytically incompetent oxidation product, 7,8-dihydrobiopterin (BH(2)). In support of this hypothesis, [(3)H]BH(4) binding studies revealed that BH(4) and BH(2) bind eNOS with equal affinity (K(d) approximately 80 nM) and BH(2) can rapidly and efficiently replace BH(4) in preformed eNOS-BH(4) complexes. Whereas the total biopterin pool of murine endothelial cells (ECs) was unaffected by 48-h exposure to diabetic glucose levels (30 mM), BH(2) levels increased from undetectable to 40% of total biopterin. This BH(2) accumulation was associated with diminished calcium ionophore-evoked NO activity and accelerated superoxide production. Since superoxide production was suppressed by NOS inhibitor treatment, eNOS was implicated as a principal superoxide source. Importantly, BH(4) supplementation of ECs (in low and high glucose-containing media) revealed that calcium ionophore-evoked NO bioactivity correlates with intracellular BH(4):BH(2) and not absolute intracellular levels of BH(4). Reciprocally, superoxide production was found to negatively correlate with intracellular BH(4):BH(2). Hyperglycemia-associated BH(4) oxidation and NO insufficiency was recapitulated in vivo, in the Zucker diabetic fatty rat model of type 2 diabetes. Together, these findings implicate diminished intracellular BH(4):BH(2), rather than BH(4) depletion per se, as the molecular trigger for NO insufficiency in diabetes.
Crabtree
Mark J
MJ
Dept. of Pharmacology, Weill Medical College of Cornell University, New York, NY 10021, USA.
Smith
Caroline L
CL
Lam
George
G
Goligorsky
Michael S
MS
Gross
Steven S
SS
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D001786:Blood Glucose / Q000378:metabolism; D002460:Cell Line; D003924:Diabetes Mellitus, Type 2 / Q000201:enzymology / Q000378:metabolism; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010084:Oxidation-Reduction; D011485:Protein Binding; D051381:Rats; D011924:Rats, Zucker; D013481:Superoxides / Q000378:metabolism*; D013997:Time Factors; D014316:Tritium
D001786:Blood Glucose; D004791:Enzyme Inhibitors; D014316:Tritium; D013481:Superoxides; D001708:Biopterins; D009569:Nitric Oxide; C017226:7,8-dihydrobiopterin; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; C496316:Nos3 protein, rat; C003402:sapropterin; D005978:Glutathione; D005947:Glucose; D019331:NG-Nitroarginine Methyl Ester