ROLE OF NITRIC OXIDE IN THE PATHOGENESIS OF LUNG DISEASE

一氧化氮是一种普遍存在的信使分子，具有重要的调节功能，由一氧化氮合酶（NOS）家族合成。已经鉴定出三种NOS异构体：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区域，一个中央钙调素结合区域，一个羧基末端还原酶结构域，带有nadph结合位点。eNOS基因位于染色体7q35-36上，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经通过其多态性变异体作为潜在的疾病标志物进行了研究。外显子7 （894 G/T）的eNOS多态性导致该蛋白的298位分别产生谷氨酸或天冬氨酸，这与心肺疾病的严重程度相关。因为谷氨酸和天冬氨酸被认为是保守的替代物，多态性被认为是基因其他功能位点的标记。我们现在在转染细胞、原代人内皮细胞和人心脏中发现，含有天冬氨酸而不是谷氨酸的eNOS在298位被切割，产生100 kda和35 kda的产物。重组或天然eNOS在裂解物（COS7）或部分纯化后通过2',5'-ADP-Sepharose和calmodulin-Sepharose进行免疫印迹检测。用羧基末端抗体进行SDS/PAGE免疫印迹显示，eNOS在135 kDa的预测位置上有一个主要的蛋白带。另外约100 kDa的条带仅存在于重组298Asp eNOS和原代细胞和G/T基因型心脏组织合成的eNOS中。使用eNOS氨基端特异性抗体，在T基因型细胞中观察到约35 kDa的免疫反应带，对应于n端残裂片段。因此，在核苷酸894处多态性的eNOS基因产生的蛋白产物对切割的敏感性不同，这表明，与先前的预测相反，这种多态性对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.