Forkert P G, D'Costa D, El-Mestrah M
Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada.
Am J Respir Cell Mol Biol. 1999 Jan;20(1):143-52. doi: 10.1165/ajrcmb.20.1.3320.
This investigation sought to establish the cellular expression and distribution of the alpha, pi, and mu classes of glutathione S-transferase (GST) enzymes in murine lung under control conditions and after treatment with tert-butyl-4-hydroxyanisole (BHA). Immunohistochemical and in situ hybridization studies were used to identify lung cells that were labeled for the GST subunits Yp, Ya, and Yb1. Immunoblotting of cytosolic proteins produced single bands of 28, 29, and 31 kD for Ya, Yp, and Yb1, respectively, in samples from untreated and BHA-treated mice. Treatment with BHA increased Ya and Yp reactivity, but this was not as marked for Yb1. Immunohistochemical staining for the Yp, Ya, and Yb1 subunits was localized in bronchioles and parenchyma of untreated and BHA-treated mice. Bronchiolar Clara and alveolar type II cells were stained to the greatest extent for all of the GST subunits. BHA treatment produced increased staining that was most pronounced in the bronchiolar epithelium. Ya and Yp were localized in the cytoplasm and nucleus, whereas Yb1 was found mainly in the cytoplasm. Immunoblots of extracted nuclear proteins revealed a band of 29 kD for Ya, with increased immunoreactivity in BHA-treated mice. In situ hybridization done with oligonucleotide probes showed abundant silver grains representing Ya, Yp, and Yb1 messenger RNA (mRNA) transcripts in the bronchioles. Grains were also localized in alveolar septa, and were most numerous in type II cells. Quantitative image analysis confirmed good agreement between relative levels of protein and mRNA transcripts. Quantities of mRNA transcripts for all subunits were increased in the parenchyma by BHA treatment, but the magnitudes of induction were most striking for Ya and Yp in the bronchioles. These results demonstrated that Ya, Yp, and Yb1 reside in specific lung areas and cells, and that in induced states, their increased expression is accompanied by increased mRNA.
本研究旨在确定在对照条件下以及用叔丁基对羟基茴香醚（BHA）处理后，小鼠肺中谷胱甘肽S-转移酶（GST）α、π和μ类酶的细胞表达及分布情况。采用免疫组织化学和原位杂交研究来鉴定标记有GST亚基Yp、Ya和Yb1的肺细胞。对未处理和BHA处理小鼠的样本进行胞质蛋白免疫印迹分析，结果显示Ya、Yp和Yb1分别产生了28、29和31kD的单条带。BHA处理增加了Ya和Yp的反应性，但对Yb1的影响不明显。未处理和BHA处理小鼠的细支气管和实质中均有Yp、Ya和Yb1亚基的免疫组织化学染色。细支气管的克拉拉细胞和肺泡II型细胞对所有GST亚基的染色程度最高。BHA处理使染色增加，在细支气管上皮中最为明显。Ya和Yp定位于细胞质和细胞核，而Yb1主要存在于细胞质中。提取的核蛋白免疫印迹显示Ya有一条29kD的条带，在BHA处理的小鼠中免疫反应性增加。用寡核苷酸探针进行的原位杂交显示，细支气管中有大量代表Ya、Yp和Yb1信使核糖核酸（mRNA）转录本的银颗粒。颗粒也定位于肺泡间隔，在II型细胞中最多。定量图像分析证实蛋白质和mRNA转录本的相对水平之间有良好的一致性。BHA处理使实质中所有亚基的mRNA转录本数量增加，但在细支气管中，Ya和Yp的诱导幅度最为显著。这些结果表明，Ya、Yp和Yb1存在于特定的肺区域和细胞中，并且在诱导状态下，它们的表达增加伴随着mRNA的增加。
