Zinchuk Vadim S, Okada Teruhiko, Akimaru Kunihiro, Seguchi Harumichi
Department of Anatomy and Cell Biology, Kochi Medical School, Nankoku, Kochi 783-8505, Japan.
Am J Physiol Gastrointest Liver Physiol. 2002 Mar;282(3):G540-8. doi: 10.1152/ajpgi.00405.2001.
In the liver, function of the bile salt export pump (Bsep), a major canalicular exporter of bile salts, is complemented by activity of the multidrug resistance protein 2 (Mrp2), a canalicular organic anions transporter. Mrp2 was found capable of transporting various anticancer drugs out of cells, eventually undermining their therapeutic potential and contributing to multidrug resistance. We employed a RT-PCR, immunoblotting, and immunofluorescence to examine their gene, protein expression, and distribution of antigenic sites in the rat liver during development from 16-day-old fetus to adult animal. Bsep mRNA was almost undetectable before birth. It was first clearly expressed in the liver of newborn rats. On the contrary, Mrp2 mRNA was seen before birth, although at low levels. In concert with mRNA expression, Bsep protein was undetectable before birth, while Mrp2 protein was already expressed. Both proteins were clearly detectable in the postnatal period. Confocal immunofluorescent microscopy showed asynchronous appearance of Bsep and Mrp2 proteins during development but their colocalization in the bile canaliculi once each one is expressed. During the gestational period, a weak immunofluorescence for Mrp2 was observed only in livers of 16-day-old embryos. No fluorescence for Bsep was seen. Both proteins were clearly visualizable after birth, although the pattern of immunostaining varied. These findings provide molecular evidence that expression of both Bsep and Mrp2 during development is transcriptionally regulated. They also point out the differences in relevance to the liver function of the systems responsible for canalicular transport of bile salts versus organic anions.
在肝脏中，胆盐输出泵（Bsep）作为主要的胆小管胆盐输出器，其功能由多药耐药蛋白2（Mrp2）的活性补充，Mrp2是一种胆小管有机阴离子转运体。研究发现Mrp2能够将多种抗癌药物转运出细胞，最终削弱其治疗潜力并导致多药耐药。我们采用逆转录聚合酶链反应（RT-PCR）、免疫印迹和免疫荧光技术，检测了从16日龄胎儿到成年动物发育过程中大鼠肝脏中它们的基因、蛋白质表达及抗原位点分布。出生前几乎检测不到Bsep mRNA。它最早在新生大鼠肝脏中清晰表达。相反，Mrp2 mRNA在出生前即可检测到，尽管水平较低。与mRNA表达一致，出生前检测不到Bsep蛋白，而Mrp2蛋白已表达。两种蛋白在出生后均可清晰检测到。共聚焦免疫荧光显微镜显示，发育过程中Bsep和Mrp2蛋白的出现不同步，但一旦各自表达，它们在胆小管中共定位。在妊娠期，仅在16日龄胚胎的肝脏中观察到Mrp2的微弱免疫荧光。未见到Bsep的荧光。出生后两种蛋白均可清晰观察到，尽管免疫染色模式有所不同。这些发现提供了分子证据，表明发育过程中Bsep和Mrp2的表达受转录调控。它们还指出了负责胆小管胆盐与有机阴离子转运的系统在肝脏功能方面的差异。
