GTP cyclohydrolase I mRNA is induced by LPS in vascular smooth muscle: characterization, sequence and relationship to nitric oxide synthase.
GTP cyclohydrolase I mRNA is induced by LPS in vascular smooth muscle: characterization, sequence and relationship to nitric oxide synthase.
复制标题
GTP 环水解酶 I mRNA 由血管平滑肌中的 LPS 诱导：特征、序列及其与一氧化氮合酶的关系。
DOI：
10.1006/bbrc.1993.2062
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发表时间：
1993
影响因子：
3.1
通讯作者：
Gross,SS
中科院分区：
文献类型：
--
作者：
Hattori,Y;Gross,SS
GTP环水解酶I(GTPCH)是合成一氧化氮合酶(NOS)的辅因子四氢生物蝶呤(BH4)的第一个限速酶。由于免疫刺激剂诱导血管平滑肌(VSM)细胞合成NO需要BH4的新合成，我们研究了免疫刺激剂是否促进GTPCH mRNA的表达。检测细菌脂多糖与干扰素-γ联合作用后大鼠血管平滑肌细胞GTPCH mRNA和BH4的表达。利用逆转录-聚合酶链式反应(RT-PCR)从大鼠肝脏GTPCH基因的已知核苷酸序列中扩增出372bp的GTPCH基因片段。该片段的双脱氧核苷酸序列分析表明，LPS/干扰素诱导的VSM GTPCH基因与肝脏GTPCH基因的同源性为100%。虽然BH4在未经处理的VSM中低于我们的检测下限，但可以检测到低水平的GTPCH mRNA。脂多糖/干扰素处理后，BH4出现，GTPCH基因表达明显增加。GTPCH信使核糖核酸(GTPCH)的表达在2 h时明显升高，4 h达到高峰，并维持在较高水平至少24 h，而放线菌酮可显著增强脂多糖/干扰素对GTPCH m RNA的诱导作用，提示一种不稳定的蛋白质抑制了GTPCH m RNA的表达。RT-PCR检测内毒素/干扰素诱导的一氧化氮合酶基因表达，发现其诱导时程与GTPCH相似。类似地，VSM暴露于脂多糖/干扰素后，胞液中一氧化氮合酶活性出现的时程与BH4含量增加的时程平行。我们的研究表明，免疫刺激剂共同诱导NOS和GTPCH基因表达：这两种事件都是VSM诱导NO合成所必需的。
GTP cyclohydrolase I (GTPCH) is the first and rate-limiting enzyme for the synthesis of tetrahydrobiopterin (BH4), a cofactor of nitric oxide synthase (NOS). As the induction of NO synthesis by immunostimulants in vascular smooth muscle (VSM) cells requiresde novosynthesis of BH4, we investigated whether immunostimulants enhance the expression of GTPCH mRNA. GTPCH mRNA and BH4 were measured in rat VSM cells after exposure to bacterial lipopolysaccharide (LPS) in combination with interferon-γ(IFN). Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify a predicted 372 bp fragment of GTPCH mRNA, deduced from the known nucleotide sequence of rat liver GTPCH cDNA. Dideoxynucleotide sequencing of the PCR fragment revealed 100% identity between LPS/IFN-induced GTPCH mRNA of VSM and the constitutive GTPCH mRNA of liver. Although BH4 was below our limit of detection in untreated VSM, low levels of GTPCH mRNA were detectable. LPS/IFN treatment triggered the appearance of BH4 and markedly increased GTPCH mRNA. Induction of GTPCH mRNA was apparent by 2 h, peaked at 4 h, and was sustained at high levels for at least 24 h. Induction of GTPCH mRNA by LPS/IFN was substantially enhanced by cycloheximide,suggesting that mRNA levels are depressed by a labile protein. Measurement of LPS/IFN-induced NOS mRNA by RT-PCR, demonstrated a timecourse of induction which mirrors that of GTPCH. Similarly, the timecourse of appearance of cytosolic NOS activity following exposure of VSM to LPS/IFN paralleled that of the increase in BH4 content. Our studies demonstrate that immunostimulants co-induce NOS and GTPCH gene expression: both events are necessary for induction of NO synthesis by VSM.