ROLE OF NITRIC OXIDE IN THE PATHOGENESIS OF LUNG DISEASE

一氧化氮是一种普遍存在的具有重要调节功能的信使分子，由一氧化氮合酶（NOS）家族合成。 已经鉴定了三种NOS同工型：两种组成型，神经元（nNOS，I型）和内皮（eNOS，III型）酶，以及一种诱导型（iNOS，II型）。 它们都有一个氨基末端血红素和精氨酸结合结构域，一个中央钙调蛋白结合区，和一个羧基末端还原酶结构域，并有一个NADPH结合位点。eNOS基因位于染色体7 q35 -36，由26个外显子组成，长度为21 kb。 鉴于NO在生理和病理生理上的重要性，eNOS在各种人类疾病的发病机制中可能发挥的作用已被研究，使用其多态性变体作为潜在的疾病marker. eNOS第7外显子（894 G/T）的多态性导致谷氨酸或天冬氨酸，分别在298位的蛋白质与心肺疾病的严重程度相关。 由于谷氨酸和天冬氨酸被认为是保守的替代，多态性被认为是基因中其他功能位点的标记。 我们现在显示，在转染的细胞，原代人内皮细胞，和人的心脏，eNOS与天冬氨酸，但不是谷氨酸，在位置298被切割，产生100-kDa和35-kDa的产品。通过在裂解物（COS 7）中或在2 '，5'-ADP-琼脂糖和钙调蛋白-琼脂糖上部分纯化后的免疫印迹来检查重组或天然eNOS。 用羧基末端抗体进行SDS/PAGE后的免疫印迹显示在eNOS的预测位置135 kDa处有一个单一的主要蛋白条带。约100 kDa的额外条带仅存在于重组298 Asp eNOS和由原代细胞和心脏组织合成的具有G/T基因型的eNOS中。 使用eNOS氨基末端特异性抗体，在约35 kDa的免疫反应性条带，对应于残留的N-末端切割片段，在这些细胞中观察到的T基因型。因此，eNOS基因与多态性在核苷酸894产生蛋白质产物具有不同的敏感性切割，这表明，在以前的预测相反，这种多态性对eNOS蛋白的功能性影响。

Nitric oxide, a ubiquitous messenger molecule with important regulatory functions, is synthesized by a family of enzymes called nitric oxide synthases (NOS). Three NOS isoforms have been identified: two constitutive, the neuronal (nNOS, type I) and endothelial (eNOS, type III) enzymes, and one inducible (iNOS, type II). All have an amino-terminal heme- and arginine-binding domain, a central calmodulin-binding region, and a carboxyl-terminal reductase domain, with an NADPH-binding site. The eNOS gene, located on chromosome 7q35-36, comprises 26 exons spanning 21 kb. In view of the physiological and pathophysiological importance of NO, the possible role of eNOS in the pathogenesis of various human diseases has been examined using its polymorphic variants as potential disease markers.An eNOS polymorphism in exon 7 (894 G/T) resulting in glutamate or aspartate, respectively, at position 298 of the protein is correlated with severity of cardiopulmonary diseases. Because glutamate and aspartate are considered to be conservative replacements, the polymorphism was thought to be a marker for a functional locus elsewhere in the gene. We now show in transfected cells, primary human endothelial cells, and human hearts, that eNOS with aspartate, but not glutamate, at position 298 is cleaved, producing 100-kDa and 35-kDa products. Recombinant or native eNOS was examined by immunoblotting either in lysates (COS7) or after partial purification over 2',5'-ADP-Sepharose and calmodulin-Sepharose. Immunoblotting after SDS/PAGE with a carboxyl-terminal antibody showed a single major protein band in the predicted position for eNOS at 135 kDa. An additional band of approximately 100 kDa was present only in the recombinant 298Asp eNOS and in the eNOS synthesized by primary cells and heart tissue with a G/T genotype. Using an eNOS amino-terminal-specific antibody, an immunoreactive band at approximately 35 kDa, corresponding to the residual N-terminal cleavage fragment, was observed in those cells with a T genotype. Thus, the eNOS gene with polymorphisms at nucleotide 894 yields protein products with differing susceptibility to cleavage, suggesting that, in contrast to prior predictions, this polymorphism has a functional effect on the eNOS protein.