P-glycoprotein expression, localization, and function in sandwich-cultured primary rat and human hepatocytes: Relevance to the hepatobiliary disposition of a model opioid peptide
P-glycoprotein expression, localization, and function in sandwich-cultured primary rat and human hepatocytes: Relevance to the hepatobiliary disposition of a model opioid peptide
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DOI：
10.1023/b:pham.0000033018.97745.0d
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发表时间：
2004-07-01
影响因子：
3.7
通讯作者：
Brouwer, KLR
中科院分区：
文献类型：
--
作者：
Hoffmaster, KA;Turncliff, RZ;Brouwer, KLR
目的。从完整肝脏中分离肝细胞涉及组织的胶原酶消化，导致细胞极化和功能载体排泄的丧失。本研究在三明治培养（SC）大鼠和人原代肝细胞中检测了复极化、p -糖蛋白（P-gp）在肝细胞小管结构域的定位以及载体转运的重建。用共聚焦显微镜和Western blotting分别测定SC肝细胞中的蛋白定位和表达。通过测定SC肝细胞胆中[d -青霉胺（2,5）]脑啡肽（H-3-DPDPE）和5（和6）-羧基-2′，7′-二氯荧光素（CDF）的排泄量来评价转运蛋白的功能。P-gp和小管标记蛋白二肽基肽酶IV（DPPIV）分别于第3天和第6天在SC大鼠肝细胞和SC人肝细胞中共定位，与光镜下可见的小管网络形成一致。多药耐药相关蛋白2 （MRP2）和P-gp在培养第6天在SC人肝细胞中共定位。P-gp的表达量随培养时间的增加而增加；MRP2在SC人肝细胞中也有类似表达。SC大鼠肝细胞中Oatp1a1的表达在培养中维持数天，而Oatp1a4的表达则下降。在SC人肝细胞中，OATP1B1的表达在第3天略有下降。OATP1B3在SC人肝细胞中表达不变。阿片肽H-3-DPDPE的体外胆汁排泄与两种动物小管蛋白的适当定位相关。SC人肝细胞中CDF的排泄证实了网络的形成和MRP2的功能。这些研究表明SC肝细胞重极化并将功能性小管转运蛋白转运到适当的细胞结构域。
Purpose. The isolation of hepatocytes from intact liver involves collagenase digestion of the tissue, resulting in loss of cell polarization and functional vectorial excretion. These studies examined repolarization, localization of P-glycoprotein (P-gp) to the canalicular domain of the hepatocyte, and re-establishment of vectorial transport in sandwich-cultured (SC) rat and human primary hepatocytes.Methods. Protein localization and expression were determined in SC hepatocytes by confocal microscopy and Western blotting, respectively. Transporter function was evaluated by measuring [D-penicillamine(2,5)] enkephalin (H-3-DPDPE) and 5 (and 6)-carboxy-2 ', 7'-dichlorofluorescein (CDF) biliary excretion in SC hepatocytes.Results. P-gp and the canalicular marker protein dipeptidyl peptidase IV(DPPIV) co-localized by Day 3 and Day 6 in SC rat hepatocytes and SC human hepatocytes, respectively, consistent with canalicular network formation visualized by light microscopy. Co-localization of multidrug resistance associated protein 2 (MRP2) and P-gp in SC human hepatocytes was observed on Day 6 in culture. Expression levels of P-gp increased slightly in both species over days in culture; similar expression was observed for MRP2 in SC human hepatocytes. Oatp1a1 expression in SC rat hepatocytes was maintained over days in culture, whereas Oatp1a4 expression decreased. OATP1B1 expression decreased slightly on Day 3 in SC human hepatocytes. OATP1B3 expression was constant in SC human hepatocytes. In vitro biliary excretion of the opioid peptide H-3-DPDPE correlated with the proper localization of canalicular proteins in both species. Excretion of CDF in SC human hepatocytes confirmed network formation and MRP2 function.Conclusions. These studies indicate that SC hepatocytes repolarize and traffic functional canalicular transport proteins to the appropriate cellular domain.