Geller D A, Lowenstein C J, Shapiro R A, Nussler A K, Di Silvio M, Wang S C, Nakayama D K, Simmons R L, Snyder S H, Billiar T R
Department of Surgery, University of Pittsburgh, PA 15261.
Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3491-5. doi: 10.1073/pnas.90.8.3491.
Nitric oxide is a short-lived biologic mediator for diverse cell types. Synthesis of an inducible nitric oxide synthase (NOS) in murine macrophages is stimulated by lipopolysaccharide (LPS) and interferon gamma. In human hepatocytes, NOS activity is induced by treatment with a combination of tumor necrosis factor, interleukin 1, interferon gamma, and LPS. We now report the molecular cloning and expression of an inducible human hepatocyte NOS (hep-NOS) cDNA. hep-NOS has 80% amino acid sequence homology to macrophage NOS (mac-NOS). Like other NOS isoforms, recognition sites for FMN, FAD, and NADPH are present, as well as a consensus calmodulin binding site. NOS activity in human 293 kidney cells transfected with hep-NOS cDNA is diminished by Ca2+ chelation and a calmodulin antagonist, reflecting a Ca2+ dependence not evident for mac-NOS. Northern blot analysis with hep-NOS cDNA reveals a 4.5-kb mRNA in both human hepatocytes and aortic smooth muscle cells following stimulation with LPS and cytokines. Human genomic Southern blots probed with human hep-NOS and human endothelial NOS cDNA clones display different genomic restriction enzyme fragments, suggesting distinct gene products for these NOS isoforms. hep-NOS appears to be an inducible form of NOS that is distinct from mac-NOS as well as brain and endothelial NOS isozymes.
一氧化氮是一种作用于多种细胞类型的短效生物介质。鼠巨噬细胞中诱导型一氧化氮合酶（NOS）的合成受脂多糖（LPS）和γ干扰素刺激。在人肝细胞中，肿瘤坏死因子、白细胞介素1、γ干扰素和LPS联合处理可诱导NOS活性。我们现在报告诱导型人肝细胞NOS（hep-NOS）cDNA的分子克隆和表达。hep-NOS与巨噬细胞NOS（mac-NOS）有80%的氨基酸序列同源性。与其他NOS同工型一样，存在黄素单核苷酸、黄素腺嘌呤二核苷酸和烟酰胺腺嘌呤二核苷酸磷酸的识别位点，以及一个共有钙调蛋白结合位点。用hep-NOS cDNA转染的人293肾细胞中的NOS活性因Ca2+螯合剂和钙调蛋白拮抗剂而降低，这反映出一种mac-NOS不明显的Ca2+依赖性。用hep-NOS cDNA进行的Northern印迹分析显示，在LPS和细胞因子刺激后人肝细胞和主动脉平滑肌细胞中均有一条4.5 kb的mRNA。用人hep-NOS和人内皮NOS cDNA克隆探测的人基因组Southern印迹显示不同的基因组限制性酶切片段，表明这些NOS同工型有不同的基因产物。hep-NOS似乎是一种诱导型NOS，与mac-NOS以及脑和内皮NOS同工酶不同。
