Phenobarbital induction of cytochromes P-450. High-level long-term responsiveness of primary rat hepatocyte cultures to drug induction, and glucocorticoid dependence of the phenobarbital response.
Phenobarbital induction of cytochromes P-450. High-level long-term responsiveness of primary rat hepatocyte cultures to drug induction, and glucocorticoid dependence of the phenobarbital response.
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苯巴比妥诱导细胞色素 P-450。
DOI：
10.1042/bj2710113
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Jauregui,HO
中科院分区：
文献类型：
--
作者：
Waxman,DJ;Morrissey,JJ;Naik,S;Jauregui,HO
在Vitrogen包被平板上无血清改良Chee培养基中培养5周，然后再暴露于PB（0.75 mM）4天的大鼠肝细胞中，研究了苯巴比妥（PB）对肝细胞色素P-450的诱导作用。免疫印迹分析表明，P-450形式PB 4（IIB 1）和PB 5（IIB 2）的诱导显着（大于50倍的增加），高达几乎一样高的水平，在PB诱导的大鼠肝脏在体内。新合成的细胞色素P-450具有酶活性，如PB诱导的肝细胞微粒体中P-450 PB 4依赖性类固醇16 β-羟化酶和戊氧基试卤灵O-脱烷基酶活性的主要诱导所示（增加高达90倍）。这些P-450的PB诱导明显增强地塞米松（50 nM-1 μ M）的存在下，这单独不是一个有效的诱导剂，并抑制了90%以上的10%胎牛血清。PB反应也被生长激素（250 ng/ml）抑制（大于85%），表明该激素可能直接作用于肝细胞，当它拮抗完整大鼠中P-450 PB 4的诱导。在未处理的肝细胞中，P-450 RLM 2（IIA 2）、P-450 3（IIA 1）和NADPH P-450还原酶水平在培养物中基本维持10-20天。后两种酶也可被PB诱导至与体内肝脏中观察到的相当的程度（3-4倍升高）。此外，P-450 c（IA 1）和P-450 3（IIA 1）是高度诱导的3-甲基胆蒽（5 μ M; 48小时曝光），即使在培养3周后。与此相反，男性特异性垂体调节的P-450型2C（IIC 11）培养肝细胞后迅速丢失，这表明补充适当的激素因子可能是必要的，其表达。目前的肝细胞培养系统表现出药物诱导剂的反应，定性和定量与在体内观察到的，并应证明有价值的PB和内源性激素的调制响应的分子和机制基础的更详细的调查。
The induction of hepatic cytochromes P-450 by phenobarbital (PB) was studied in rat hepatocytes cultured for up to 5 weeks on Vitrogen-coated plates in serum-free modified Chee's medium then exposed to PB (0.75 mM) for an additional 4 days. Immunoblotting analysis indicated that P-450 forms PB4 (IIB1) and PB5 (IIB2) were induced dramatically (greater than 50-fold increase), up to levels nearly as high as those achieved in PB-induced rat liver in vivo. The newly synthesized cytochrome P-450 was enzymically active, as shown by the major induction of the P-450 PB4-dependent steroid 16 β-hydroxylase and pentoxyresorufin O-dealkylase activities in the PB-induced hepatocyte microsomes (up to 90-fold increase). PB induction of these P-450s was markedly enhanced by the presence of dexamethasone (50 nM-1 microM), which alone was not an affective inducing agent, and was inhibited by greater than 90% by 10% fetal bovine serum. The PB response was also inhibited (greater than 85%) by growth hormone (250 ng/ml), indicating that this hormone probably acts directly on the hepatocyte when it antagonizes the induction of P-450 PB4 in intact rats. In untreated hepatocytes, P-450 RLM2 (IIA2), P-450 3 (IIA1) and NADPH P-450 reductase levels were substantially maintained in the cultures for 10-20 days. The latter two enzymes were also inducible by PB to an extent (3-4 fold elevation) that is comparable with that observed in the liver in vivo. Moreover, P-450c (IA1) and P-450 3 (IIA1) were highly inducible by 3-methylcholanthrene (5 microM; 48 h exposure) even after 3 weeks in culture. In contrast, the male-specific pituitary-regulated P-450 form 2c (IIC11) was rapidly lost upon culturing the hepatocytes, suggesting that supplementation of appropriate hormonal factors may be necessary for its expression. The present hepatocyte culture system exhibits a responsiveness to drug inducers that is qualitatively and quantitatively comparable with that observed in vivo, and should prove valuable for more detailed investigations of the molecular and mechanistic basis of the response to PB and its modulation by endogenous hormones.