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。已经鉴定出三种一氧化氮合酶亚型：两种构成酶，神经型(nNOS，I型)和内皮型(eNOS，III型)，以及一种诱导型(iNOS，II型)。它们都有一个氨基末端的血红素和精氨酸结合结构域，一个中央的钙调蛋白结合区，以及一个带有NADPH结合位点的羧基末端还原酶结构域。ENOS基因位于染色体7q35-36，由26个外显子组成，全长21kb。鉴于NO在生理和病理生理学上的重要性，人们利用eNOS的多态变异作为潜在的疾病标志物，研究了eNOS在人类各种疾病发病机制中的可能作用。eNOS基因外显子7(894G/T)上298位分别产生谷氨酸和天冬氨酸的eNOS多态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品，这种多态被认为是该基因其他地方的一个功能位点的标记。我们现在发现，在转基因细胞、原代人内皮细胞和人心脏中，eNOS在第298位含有天冬氨酸，而不是谷氨酸，被切割，产生100 kDa和35 kDa的产物。免疫印迹法检测重组或天然eNOS在裂解产物(COS7)中或经2‘，5’-ADP-琼脂糖凝胶和钙调蛋白琼脂糖凝胶部分纯化后的表达。用羧基末端抗体进行SDS/PAGE免疫印迹显示，eNOS在135 kDa的预测位置有一条单一的主蛋白条带。另一条约100 kDa的条带仅存在于重组的298Asp eNOS以及由G/T基因型的原代细胞和心脏组织合成的eNOS中。使用eNOS氨基末端特异性抗体，在T基因型的细胞中观察到一条约35 kDa的免疫反应条带，对应于残留的N端裂解片段。因此，eNOS基因894位核苷酸的多态产生不同的切割敏感性的蛋白质产物，这表明与先前的预测相反，这种多态对eNOS蛋白具有功能影响。

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.