Gross S S, Levi R
Department of Pharmacology, Cornell University Medical College, New York, New York 10021.
J Biol Chem. 1992 Dec 25;267(36):25722-9.
Nitric oxide (NO) synthesis is induced in vascular smooth muscle cells by lipopolysaccharide (LPS) where it appears to mediate a variety of vascular dysfunctions. In some cell types tetrahydrobiopterin (BH4) synthesis has also been found to be induced by cytokines. Because BH4 is a cofactor for NO synthase, we investigated whether BH4 synthesis is required for LPS-induced NO production in rat aortic smooth muscle cells (RASMC). The total biopterin content (BH4 and more oxidized states) of untreated RASMC was below our limit of detection. However, treatment with LPS caused a significant rise in biopterin levels and an induction of NO synthesis; both effects of LPS were markedly potentiated by interferon-gamma. 2,4-Diamino-6-hydroxypyrimidine (DAHP), a selective inhibitor of GTP cyclohydrolase I, the rate-limiting enzyme for de novo BH4 synthesis, completely abolished the elevated biopterin levels induced by LPS. DAHP also caused a concentration-dependent inhibition of LPS-induced NO synthesis. Inhibition of NO synthesis by DAHP was reversed by sepiapterin, an agent which circumvents the inhibition of biopterin synthesis by DAHP by serving as a substrate for BH4 synthesis via the pterin salvage pathway. The reversal by sepiapterin was overcome by methotrexate, an inhibitor of the pterin salvage pathway. Sepiapterin, and to a lesser extent BH4, dose-dependently enhanced LPS-induced NO synthesis, indicating that BH4 concentration limits the rate of NO production by LPS-activated RASMC. Sepiapterin also caused LPS-induced NO synthesis to appear with an abbreviated lag period phase, suggesting that BH4 availability also limits the onset of NO synthesis. In contrast to the stimulation of LPS-induced NO synthesis, observed when sepiapterin was given alone, sepiapterin became a potent inhibitor of NO synthesis in the presence of methotrexate. This is attributable to a direct inhibitory action of sepiapterin on GTP cyclohydrolase I, an activity which is only revealed after blocking the metabolism of sepiapterin to BH4. Further studies with sepiapterin, methotrexate, and N-acetylserotonin (an inhibitor of the BH4 synthetic enzyme, sepiapterin reductase) indicated that the BH4 is synthesized in RASMC predominantly from GTP; however, a lesser amount may derive from pterin salvage. We demonstrate that BH4 synthesis is an absolute requirement for induction of NO synthesis by LPS in vascular smooth muscle. Our findings also suggest that pterin synthesis inhibitors may be useful for the therapy of endotoxin- and cytokine-induced shock.
脂多糖（LPS）可诱导血管平滑肌细胞合成一氧化氮（NO），而NO似乎介导了多种血管功能障碍。在某些细胞类型中，还发现细胞因子可诱导四氢生物蝶呤（BH4）的合成。由于BH4是一氧化氮合酶的辅因子，我们研究了在大鼠主动脉平滑肌细胞（RASMC）中，LPS诱导的NO产生是否需要BH4的合成。未处理的RASMC的总生物蝶呤含量（BH4和更多氧化态）低于我们的检测限。然而，LPS处理导致生物蝶呤水平显著升高并诱导NO合成；干扰素-γ可显著增强LPS的这两种作用。2,4-二氨基-6-羟基嘧啶（DAHP）是GTP环水解酶I的选择性抑制剂，GTP环水解酶I是从头合成BH4的限速酶，它完全消除了LPS诱导的生物蝶呤水平升高。DAHP还导致LPS诱导的NO合成呈浓度依赖性抑制。DAHP对NO合成的抑制作用可被蝶酰谷氨酸逆转，蝶酰谷氨酸通过蝶呤补救途径作为BH4合成的底物，从而规避了DAHP对生物蝶呤合成的抑制。甲氨蝶呤是蝶呤补救途径的抑制剂，可克服蝶酰谷氨酸的逆转作用。蝶酰谷氨酸以及程度较轻的BH4可剂量依赖性地增强LPS诱导的NO合成，表明BH4浓度限制了LPS激活的RASMC产生NO的速率。蝶酰谷氨酸还使LPS诱导的NO合成出现的延迟期缩短，这表明BH4的可利用性也限制了NO合成的起始。与单独给予蝶酰谷氨酸时观察到的对LPS诱导的NO合成的刺激作用相反，在存在甲氨蝶呤的情况下，蝶酰谷氨酸成为NO合成的有效抑制剂。这归因于蝶酰谷氨酸对GTP环水解酶I的直接抑制作用，这种活性只有在阻断蝶酰谷氨酸向BH4的代谢后才会显现出来。使用蝶酰谷氨酸、甲氨蝶呤和N-乙酰血清素（BH4合成酶蝶酰谷氨酸还原酶的抑制剂）进行的进一步研究表明，RASMC中的BH4主要由GTP合成；然而，较少部分可能来自蝶呤补救途径。我们证明BH4的合成是LPS诱导血管平滑肌细胞合成NO的绝对必要条件。我们的研究结果还表明，蝶呤合成抑制剂可能对内毒素和细胞因子诱导的休克治疗有用。
