Expression of cytochrome P450 3A in amphibian, rat, and human kidney.
Expression of cytochrome P450 3A in amphibian, rat, and human kidney.
复制标题
细胞色素 P450 3A 在两栖动物、大鼠和人肾中的表达。
DOI：
10.1016/0003-9861(92)90159-t
复制
发表时间：
1992
影响因子：
3.9
通讯作者：
Watlington,CO
中科院分区：
文献类型：
--
作者：
Schuetz,EG;Schuetz,JD;Grogan,WM;Naray-Fejes-Toth,A;Fejes-Toth,G;Raucy,J;Guzelian,P;Gionela,K;Watlington,CO
家族3A哺乳动物肝细胞色素P450（3A 1，大鼠; 3A 3/4，人）催化内源性类固醇的6β-羟基化，并且是类固醇诱导型。我们最近发现A6细胞（蟾蜍肾上皮细胞系）含有皮质酮6β-羟化酶活性作为类固醇诱导的微粒体细胞色素P450，提出了A6细胞中皮质酮6β-羟化酶活性由3A家族成员催化的可能性。我们发现，地塞米松诱导细胞的A6细胞微粒体与抗3A家族蛋白的抗体孵育可特异性抑制皮质酮6β-羟化酶活性。在用家族3A特异性抗体显影的免疫印迹上分析的来自A6细胞的微粒体揭示了免疫反应性蛋白，并且用皮质酮或地塞米松处理A6增加了3A免疫反应性蛋白的量。此外，A6 RNA在北方印迹上与3A cDNA杂交，来自A6细胞的基因组DNA在南方印迹上与3A cDNA杂交。因此，蟾蜍肾A6细胞表达的家族3A P450是免疫化学，功能和遗传相关的哺乳动物肝脏3A蛋白。在两栖动物肾脏中的这些发现证实，我们检查了哺乳动物肾脏家族3A蛋白的证据。正常成年大鼠肾脏冷冻切片与3A 1抗体孵育的免疫细胞化学研究显示，免疫反应性只与集合管。人肾微粒体的免疫印迹分析发现三条蛋白带，分别代表3A 3/4、3A 5和一个53-kDaMr蛋白，与人3A抗体免疫反应。一个意想不到的发现是3A 3/4在人肾脏中的多态性表达，其中所测试的7个成人肾脏中仅一个（14%）表达该蛋白质，而3A 5（一种在成人肝脏中多态性表达的蛋白质）通常存在于所测试的成人肾脏样品中。由于人胎肝含有3A P450家族，我们通过用人肝3A抗体进行免疫印迹分析来检查人胎肾微粒体，并发现初步鉴定为3A 7的蛋白质的表达。因此，与A6两栖动物细胞一样，家族3A P450蛋白和mRNA是哺乳动物（包括人）肾脏中的主要功能组分。
Family 3A mammalian liver cytochromes P450 (3A1, rat; 3A3/4, human) catalyze the 6β-hydroxylation of endogenous steroids and are steroid inducible. Our recent finding that A6 cells (a toad kidney epithelial cell line) contain corticosterone 6β-hydroxylase activity as a steroid-inducible microsomal cytochrome P450 raised the possibility that corticosterone 6β-hydroxylase activity in the A6 cells is catalyzed by a member of the 3A family. We found that incubation of A6 cell microsomes from dexamethasone-induced cells with antibodies against family 3A proteins specifically inhibited corticosterone 6β-hydroxylase activity. Microsomes from A6 cells analyzed on immunoblots developed with family 3A specific antibodies revealed immunoreactive proteins and treatment of A6 with corticosterone or dexamethasone increased the amounts of 3A immunoreactive protein(s). Furthermore, A6 RNA hybridized with 3A cDNAs on Northern blots and genomic DNA from A6 cells hybridized with a 3A cDNA on a Southern blot. Thus, toad kidney A6 cells express a family 3A P450 that is immunochemically, functionally, and genetically related to the mammalian liver 3A proteins. Prompted by these findings in amphibian kidney, we examined mammalian kidney for evidence of family 3A proteins. Immunocytochemical studies of frozen cryostat sections of normal adult rat kidney incubated with 3A1 antibody showed immunoreactivity only with collecting duct. Immunoblot analysis of human kidney microsomes found three protein bands representing 3A3/4, 3A5, and a 53-kDaMrprotein immunoreactive with human 3A antibody. An unexpected finding was the polymorphic expression of 3A3/4 in human kidney with only one of seven (14%) adult human kidneys tested expressing this protein while 3A5, a protein which is polymorphically expressed in adult human livers, was routinely present in the adult human kidney samples tested. Since human fetal liver contains a family 3A P450 we examined human fetal kidney microsomes by immunoblot analysis with human liver 3A antibody and found expression of a protein tentatively identified as 3A7. Thus, like A6 amphibian cells, family 3A P450 proteins and mRNAs are prominent, functional components in the kidney of mammals, including man.