Serabjit-Singh C J, Nishio S J, Philpot R M, Plopper C G
National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Mol Pharmacol. 1988 Mar;33(3):279-89.
The cytochrome P-450 monooxygenase system of the mammalian lung is known to be associated with the microsomal subcellular fraction and has been demonstrated in two pulmonary cell types rich in endoplasmic reticulum: Clara cells and type II pneumocytes. However, analysis of ultracellular fractions, isolated cell preparations, or light microscopic immunohistochemical studies of tissue sections has permitted only limited resolution of the distribution of this enzyme system within the 40 or more cell types of the lung. Therefore, we have used the greater resolving power of transmission electron microscopy and immunogold labeling to characterize the cellular and subcellular distribution of the cytochrome P-450 system in the lung. In Lowicryl-embedded sections of lung from adult rabbits, antisera (1:10,000) against the constitutive pulmonary microsomal cytochrome P-450 monooxygenase isozymes 2 and 5 and NADPH-cytochrome P-450 reductase (anti-2, anti-5 and anti-R) bound specifically to regions known to be rich in agranular endoplasmic reticulum (AER) in the cytoplasm of Clara cells. The plasma membranes of bronchiolar Clara cells, the tips of microvillae of ciliated cells, secretory granules of goblet cells, and the cell membrane and pinocytotic vesicles of endothelial cells were all intensely labeled with anti-2 and anti-5 but not with anti-R, even at a 10-fold higher concentration. The intensity of labeling of AER in Clara cells with anti-R and anti-2, but not anti-5, appeared to correlate positively with the cellular content of secretory granules. The Golgi membranes of ciliated cells were labeled intensely with anti-5 only. The plasma membrane of type II pneumocytes was not labeled by any of the antisera, but with anti-2 or anti-5 there was labeling of AER-associated vacuoles, the membranous residue of lamellar bodies, and, to some extent, mitochondria; at 1:5,000 but not 1:10,000 dilution, staining with anti-R was qualitatively similar. Type I pneumocytes, ciliated cell cytoplasm, and nuclei were essentially unlabeled. Immunoblots (Western) of tracheal homogenates yielded no evidence for epitopes other than those in microsomal fractions from whole lung. Contact blots of fresh whole trachea, before but not after lavage, bound anti-2 and anti-R. Thus, we have demonstrated for the first time that components of the pulmonary cytochrome P-450 monooxygenase, although localized in the AER-rich regions of the Clara cells and type II pneumocytes, are not restricted to these cell types or to the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 400 WORDS)
哺乳动物肺中的细胞色素P-450单加氧酶系统已知与微粒体亚细胞部分相关，并且已在富含内质网的两种肺细胞类型中得到证实：克拉拉细胞和II型肺细胞。然而，对超细胞部分、分离的细胞制剂或组织切片的光学显微镜免疫组织化学研究，仅能有限地分辨该酶系统在肺的40多种细胞类型中的分布。因此，我们利用透射电子显微镜和免疫金标记的更高分辨率，来表征肺中细胞色素P-450系统的细胞和亚细胞分布。在成年兔肺的Lowicryl包埋切片中，针对组成型肺微粒体细胞色素P-450单加氧酶同工酶2和5以及NADPH-细胞色素P-450还原酶的抗血清（1:10,000）（抗-2、抗-5和抗-R）特异性结合到克拉拉细胞胞质中已知富含无颗粒内质网（AER）的区域。细支气管克拉拉细胞的质膜、纤毛细胞微绒毛尖端、杯状细胞的分泌颗粒以及内皮细胞的细胞膜和胞饮小泡，即使在浓度高10倍的情况下，也都被抗-2和抗-5强烈标记，但未被抗-R标记。用抗-R和抗-2而非抗-5标记克拉拉细胞中AER的强度，似乎与分泌颗粒的细胞含量呈正相关。纤毛细胞的高尔基体膜仅被抗-5强烈标记。II型肺细胞的质膜未被任何一种抗血清标记，但用抗-2或抗-5标记时，AER相关的液泡、板层小体的膜残余物以及在一定程度上的线粒体有标记；在1:5,000而非1:10,000稀释度下，抗-R染色在质量上相似。I型肺细胞、纤毛细胞胞质和细胞核基本未被标记。气管匀浆的免疫印迹（Western）未提供除全肺微粒体部分以外的表位证据。新鲜全气管的接触印迹，在灌洗前而非灌洗后，结合抗-2和抗-R。因此，我们首次证明，肺细胞色素P-450单加氧酶的成分，尽管定位于克拉拉细胞和II型肺细胞富含AER的区域，但并不局限于这些细胞类型或内质网。（摘要截短于400字）
