Divergent effects of cycloheximide on the induction of class II and class III cytochrome P450 mRNAs in cultures of adult rat hepatocytes.
Divergent effects of cycloheximide on the induction of class II and class III cytochrome P450 mRNAs in cultures of adult rat hepatocytes.
复制标题
放线菌酮对成年大鼠肝细胞培养物中 II 类和 III 类细胞色素 P450 mRNA 诱导的不同影响。
DOI：
10.1016/0003-9861(90)90433-y
复制
发表时间：
1990
影响因子：
3.9
通讯作者：
Guzelian,PS
中科院分区：
文献类型：
--
作者：
Burger,HJ;Schuetz,EG;Schuetz,JD;Guzelian,PS
我们之前报道过，当从成年雄性大鼠分离的肝细胞在无血清培养基中培养于基质凝胶（一种重组基底膜凝胶）上时，苯巴比妥治疗可能会刺激II类细胞色素P450b/e和III类细胞色素P450p的基因表达（e . G. Schuetzet al., 1990）。医学杂志。Chem.265, 1188 - 1192)。在本研究中，对这些细胞色素mRNA增加所需蛋白质合成的研究表明，在向培养基中添加苯巴比妥或“苯巴比妥样”诱导剂之前，用环己亚胺对细胞进行预处理，抑制了P450b/e mRNA的诱导（46-90%），而P450p mRNA的积累则增强了（2- 19倍）。血红素消耗似乎不能解释这些观察结果，因为环己亚胺对P450b/e mRNA诱导的抑制作用不能通过补充外源血红素或δ-氨基乙酰丙酸来克服。由于IIIA类P450s受性别和苯巴比妥的调节，我们在雄性大鼠肝细胞培养物中检测了P450p mRNA的基础表达，发现环己亚胺治疗没有效果。然而，在从雌性大鼠分离的肝细胞培养中，P450p mRNA几乎检测不到，环己亚胺处理大大增强了P450p mRNA的表达。正如在培养细胞中观察到的那样，用环己亚胺处理活的雌性大鼠也将P450p mRNA的数量增加到与未处理的雄性大鼠肝脏中的水平相当。分别与p450pcn1iiia1和P450PCN2（IIIA2）特异性寡核苷酸杂交的Northern blots分析显示，未经处理的雄性大鼠肝脏和从这些动物制备的肝细胞培养物表达了可检测量的P450PCN1(IIIA1)mRNA。这些分析证实，环己亚胺处理选择性地增加了雌性大鼠肝脏中的P450PCN1(IIIA1)mRNA，而与之密切相关的雄性特异性家族成员P450PCN2（IIIA2）的mRNA量不受影响。我们得出结论，苯巴比妥诱导P450b/e和P450p的途径，以及P450PCN1（IIIA1）和P450PCN2（IIIA2）的性别特异性基础表达途径并不相同，可以通过它们对正在进行的蛋白质合成抑制的不同反应来区分。
We have previously reported that when hepatocytes isolated from adult male rats are cultured in serum-free medium on matrigel, a reconstituted basement membrane gel, it is possible to elicit a stimulation of gene expression for both Class II cytochrome P450b/e and Class III cytochrome P450p by phenobarbital treatment (E. G. Schuetzet al., 1990J. Biol. Chem.265, 1188–1192). In the present study, an investigation of the requirement of protein synthesis for the rise in mRNAs for these cytochromes, pretreatment of the cells with cycloheximide prior to adding phenobarbital or “phenobarbital-like” inducers to the culture medium inhibited induction of P450b/e mRNA (46–90%), whereas the accumulation of P450p mRNA was enhanced (2- to 19-fold). Heme depletion did not appear to explain these observations because the inhibitory effects of cycloheximide on the induction of P450b/e mRNA were not overcome by supplementation of the medium with exogenous heme or with δ-aminolevulinic acid. Because Class IIIA P450s are regulated by gender as well as by phenobarbital, we examined the basal expression of P450p mRNA in cultures of hepatocytes derived from male rats and found that cycloheximide treatment was without effect. However, in cultures of hepatocytes isolated from female rats, where P450p mRNA is barely detectable, cycloheximide treatment greatly enhanced expression of P450p mRNA. As was observed in the cultured cells, the treatment of living female rats with cycloheximide also increased the amounts of P450p mRNA to levels comparable to those found in livers of untreated male rats. Analysis of Northern blots hybridized with oligonucleotides specific for P450PCN1IIIA1and P450PCN2(IIIA2), respectively, revealed that untreated male rat liver and cultures of hepatocytes prepared from these animals expressed readily detectable amounts of P450PCN1(IIIA1)mRNA. Such analyses confirmed that cycloheximide treatment selectively increased P450PCN1(IIIA1)mRNA in female rat liver, whereas the amount of mRNA for P450PCN2(IIIA2), a closely related male-specific family member, was unaffected. We conclude that the pathways for the induction of P450b/e and P450p by phenobarbital, and the pathways for the gender-specific basal expression of P450PCN1(IIIA1)and P450PCN2(IIIA2)are not the same and can be distinguished by their differential response to inhibition of ongoing protein synthesis.