Goodwin B, Liddle C, Murray M, Tapner M, Rooney T, Farrell G C
Department of Clinical Pharmacology, Storr Liver Unit, University of Sydney, Westmead Hospital, NSW, Australia.
Biochem Pharmacol. 1996 Jul 26;52(2):219-27. doi: 10.1016/0006-2952(96)00179-7.
Hepatocytes cultured on matrigel express many liver-specific functions, but the levels and activities of the predominant male-specific rat hepatic CYPs, 3A2 and 2C11, decline rapidly in culture. Metyrapone maintains the level of total cytochrome P450 of rat hepatocytes in primary culture, but the mechanism underlying this effect has not been completely elucidated. The present study sought to determine whether metyrapone acts solely to stabilise CYP proteins in rat hepatocytes cultured on matrigel, or whether it also influences mRNA levels of the encoding genes. Metyrapone maintained the level of total cytochrome P450 in cultured hepatocytes so that values were > 200% of those found in untreated control cells 24 hr after isolation. At this time, CYP3A2-mediated testosterone 6 beta-hydroxylation was approximately 7-fold higher in hepatocytes cultured in the presence of metyrapone than in control cells, and CYP2C11-dependent testosterone 2 alpha- and 16 alpha-hydroxylation activities were between 2 and 3-fold greater. The results inferred from catalytic activities were supported by immunoquantitation of CYP3A and 2C11 proteins. The trend of increased CYP protein levels in metyrapone-treated cells continued throughout the 48-hr culture period. In control cells, CYP3A2 and 2C11 mRNA levels fell abruptly in culture to reach values at 24 hr that were < 30% of those in freshly isolated cells; addition of metyrapone failed to arrest this fall. However, treatment of cells with metyrapone considerably elevated levels of one or more CYP3A subfamily mRNA species, as detected by a riboprobe based on the cDNA for CYP3A1 ("CYP3A1-like mRNA') that were demonstrated, by another riboprobe, not to be CYP3A2 or RNCYP3AM. RT-PCR of mRNA prepared from cultured hepatocytes, followed by restriction mapping of the cloned cDNAs was used to characterise the CYP3A induced by metyrapone. This revealed that elevated levels of the CYP3A1-like mRNA were attributable to induction of RL33/cDEX mRNA; there were no CYP3A1 cDNAs isolated from these cells. These data are interpreted as indicating that metyrapone stabilises the expression of cytochrome P450 in culture by both pre- and posttranslational mechanisms. The particular mechanism employed is gene-specific, whereby even the highly homologous genes CYP3A2, RL33/cDEX and, possibly, RNCYP3AM are subject to different types of regulation in the presence of metyrapone.
在基质胶上培养的肝细胞可表达多种肝脏特异性功能，但雄性大鼠肝脏中主要的细胞色素P450同工酶3A2和2C11的水平及活性在培养过程中迅速下降。甲吡酮可维持原代培养大鼠肝细胞中细胞色素P450的总量水平，但其作用机制尚未完全阐明。本研究旨在确定甲吡酮是否仅作用于稳定在基质胶上培养的大鼠肝细胞中的细胞色素P450蛋白，还是也会影响编码基因的mRNA水平。甲吡酮维持了培养肝细胞中细胞色素P450的总量水平，使得分离后24小时的值是未处理对照细胞中值的200%以上。此时，在甲吡酮存在下培养的肝细胞中，CYP3A2介导的睾酮6β-羟化作用比对照细胞中高约7倍，CYP2C11依赖的睾酮2α-和16α-羟化活性高2至3倍。催化活性推断出的结果得到了CYP3A和2C11蛋白免疫定量的支持。在整个48小时的培养期内，甲吡酮处理的细胞中CYP蛋白水平持续升高。在对照细胞中，CYP3A2和2C11的mRNA水平在培养过程中急剧下降，到24小时时降至新鲜分离细胞中水平的30%以下；添加甲吡酮未能阻止这种下降。然而，用甲吡酮处理细胞可显著提高一种或多种CYP3A亚家族mRNA种类的水平，如用基于CYP3A1 cDNA的核糖探针检测到的（“CYP3A1样mRNA”），另一种核糖探针证明其不是CYP3A2或RNCYP3AM。从培养的肝细胞制备的mRNA进行RT-PCR，随后对克隆的cDNA进行限制性图谱分析，以表征甲吡酮诱导的CYP3A。这表明CYP3A1样mRNA水平升高归因于RL33/cDEX mRNA的诱导；从这些细胞中未分离到CYP3A1 cDNA。这些数据被解释为表明甲吡酮通过翻译前和翻译后机制稳定培养中的细胞色素P450表达。所采用的具体机制是基因特异性的，即在甲吡酮存在下，即使是高度同源的基因CYP3A2、RL33/cDEX以及可能的RNCYP3AM也受到不同类型的调控。
