Chichester C H, Philpot R M, Weir A J, Buckpitt A R, Plopper C G
School of Veterinary Medicine, Department of Anatomy, University of California, Davis 95616.
Am J Respir Cell Mol Biol. 1991 Feb;4(2):179-86. doi: 10.1165/ajrcmb/4.2.179.
The nonciliated bronchiolar epithelial (Clara) cell of the mouse is highly susceptible to toxicants that undergo metabolic activation, presumably because this cell type has high levels of cytochrome P-450 monooxygenases. As a first step in further defining the role of Clara cells in pulmonary xenobiotic activation and detoxication, we have isolated Clara cells (75 to 80% purity) and characterized them morphologically and biochemically. The identity of Clara cells, confirmed by transmission electron microscopy, was based on several features, including abundant agranular endoplasmic reticulum, large mitochondria, and dense secretory granules. Immunocytochemistry of isolated mouse cells showed that the majority were positive with antibodies against three major components of the pulmonary cytochrome P-450 monooxygenase system, cytochrome P-450 isozymes 2 (IIB), 5 (IVB), and NADPH cytochrome P-450 reductase, purified from rabbit lung. The isolated cells also showed a positive reaction with an antibody against the cytochrome P-450 isozyme that is active in the stereoselective metabolism of naphthalene, cytochrome P-450 mN (mN). Immunocytochemistry using the antibody against cytochrome P-450 isozyme 6 (IA1), purified from rabbit lung, showed no reaction in the isolated cells. The presence of intact cytochrome P-450 protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blots of homogenates of isolated cell preparations. The N-demethylation of benzphetamine and epoxidation of naphthalene occurred at easily measurable rates in incubations of isolated Clara cells. In contrast, diols, quinones, and monohydroxylated benzo(a)pyrene metabolites, analyzed by high performance liquid chromatography, were undetectable in extracts of Clara cells incubated with 3H-labeled substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
小鼠的无纤毛细支气管上皮（克拉拉）细胞对经历代谢活化的毒物高度敏感，推测是因为这种细胞类型具有高水平的细胞色素P-450单加氧酶。作为进一步确定克拉拉细胞在肺部异生物质活化和解毒中作用的第一步，我们分离了克拉拉细胞（纯度为75%至80%），并对其进行了形态学和生物化学特征分析。通过透射电子显微镜确认的克拉拉细胞的身份基于几个特征，包括丰富的无颗粒内质网、大线粒体和密集的分泌颗粒。对分离的小鼠细胞进行免疫细胞化学分析表明，大多数细胞对针对从兔肺中纯化的肺细胞色素P-450单加氧酶系统的三个主要成分、细胞色素P-450同工酶2（IIB）、5（IVB）和NADPH细胞色素P-450还原酶的抗体呈阳性反应。分离的细胞对针对在萘的立体选择性代谢中具有活性的细胞色素P-450同工酶、细胞色素P-450 mN（mN）的抗体也呈阳性反应。使用从兔肺中纯化的针对细胞色素P-450同工酶6（IA1）的抗体进行免疫细胞化学分析表明，分离的细胞中无反应。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳和分离细胞制剂匀浆的蛋白质免疫印迹法证实了完整细胞色素P-450蛋白的存在。在分离的克拉拉细胞孵育过程中，苄非他明的N-去甲基化和萘的环氧化以易于测量的速率发生。相比之下，通过高效液相色谱分析的二醇、醌和单羟基化苯并(a)芘代谢物在与3H标记底物孵育的克拉拉细胞提取物中未检测到。（摘要截短于250字）
