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
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
复制标题
DOI：
10.1152/ajpheart.00823.2007
复制
发表时间：
2008-04-01
影响因子：
4.8
通讯作者：
Gross, Steven S.
中科院分区：
文献类型：
--
作者：
Crabtree, Mark J.;Smith, Caroline L.;Gross, Steven S.
5，6，7，8-四氢生物蝶呤(BH4)是一氧化氮合酶(NOSS)的重要辅因子。在糖尿病和其他慢性血管炎症条件下，BH4的氧化可导致辅因子不足和内皮型一氧化氮合酶(ENOS)解偶联，表现为从一氧化氮(NO)到超氧化物的产生。在这里，我们检验了eNOS解偶联不仅仅是BH4不足的结果，而是BH4与其催化不能的氧化产物7，8-二氢生物蝶呤(BH2)比率降低的结果。为了支持这一假设，[H-3]BH4结合研究表明，BH4和BH2以相同的亲和力结合eNOS(K-d约为80 nM)，BH2可以快速有效地取代预先形成的eNOS-BH4复合体中的BH4。虽然小鼠内皮细胞(ECs)的总生物蝶呤水平不受糖尿病血糖水平(30 MM)暴露48小时的影响，但BH2水平从无法检测到的水平增加到总生物蝶呤水平的40%。这种BH2的积累与钙离子载体诱发的NO活性降低和超氧化物生成加速有关。由于超氧化物歧化酶的产生被一氧化氮合酶抑制剂抑制，内皮型一氧化氮合酶被认为是主要的超氧化物歧化酶来源。重要的是，BH4补充内皮细胞(在低糖和高糖介质中)显示，钙离子载体引起的NO生物活性与细胞内BH4：BH2相关，而不是细胞内BH4的绝对水平。反过来，超氧化物的产生被发现与细胞内的BH4：BH2负相关。在2型糖尿病的Zucker糖尿病肥胖大鼠模型中，体内再现了与高血糖相关的BH4氧化和NO不足。总之，这些发现暗示细胞内BH4：BH2的减少，而不是BH4本身的消耗，是糖尿病患者无功能不全的分子触发因素。
5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthases (NOSs). Oxidation of BH4, 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 BH4 insufficiency, but rather results from a diminished ratio of BH4 vs. its catalytically incompetent oxidation product, 7,8-dihydrobiopterin (BH2). In support of this hypothesis, [H-3] BH4 binding studies revealed that BH4 and BH2 bind eNOS with equal affinity (K-d approximate to 80 nM) and BH2 can rapidly and efficiently replace BH4 in preformed eNOS-BH4 complexes. Whereas the total biopterin pool of murine endothelial cells (ECs) was unaffected by 48-h exposure to diabetic glucose levels (30 mM), BH2 levels increased from undetectable to 40% of total biopterin. This BH2 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, BH4 supplementation of ECs (in low and high glucose-containing media) revealed that calcium ionophore-evoked NO bioactivity correlates with intracellular BH4: BH2 and not absolute intracellular levels of BH4. Reciprocally, superoxide production was found to negatively correlate with intracellular BH4: BH2. Hyperglycemia-associated BH4 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 BH4: BH2, rather than BH4 depletion per se, as the molecular trigger for NO insufficiency in diabetes.