Kwon Y, Kamath A V, Morris M E
Department of Pharmaceutics, School of Pharmacy, State University of New York at Buffalo, Amherst 14260, USA.
J Pharm Sci. 1996 Sep;85(9):935-9. doi: 10.1021/js9600540.
P-glycoprotein (P-gp), the multidrug resistance (MDR) gene product, is exclusively located on the canalicular membrane of hepatocytes. Recent studies using isolated rat canalicular liver plasma membrane (cLPM) vesicles indicate that daunomycin (DNM) is a substrate for the ATP-dependent P-gp efflux system in the rat liver. The isoforms of P-gp present in cLPM and in cancer cell lines differ in that the major form present in the liver represents the gene product of mdr2 in mice (MDR3 in humans; class III) while the isoform of P-gp in cancer cells is the gene product of mdr1 in mice (MDR1 in humans, class I). The objective of this study was to examine the inhibitory effects of various organic compounds, most of which have been studied previously in MDR cancer cells, on P-gp-mediated [3H]DNM uptake into cLPM. Also, the stereospecificity of P-gp for its substrates was investigated by comparing the inhibitory effects of the enantiomers and the racemic mixtures of verapamil and propranolol. DNM exhibited ATP-dependent active transport into rat liver cLPM with a Km of 26.8 +/- 13.4 microM and a Vmax of 4.9 +/- 0.8 nmol/45 s/mg of protein (n = 4). ADP, AMP, and a nonhydrolyzable ATP analogue did not increase DNM transport over the control value. Thirty-one potential inhibitors were examined; only acridine orange, doxorubicin, verapamil, propranolol, phosphatidylcholine, beta-estradiol glucuronide, and DNM itself showed statistically significant inhibition of [3H]DNM uptake into cLPM. These results suggest that only a limited number of substrates bind to or are transported across the hepatic canalicular membrane via P-gp. Phosphatidylcholine, a substrate for the gene product of the class III P-gp gene, produced significant inhibition of [3H]DNM transport (30.6% at a 10-fold-higher substrate concentration), suggesting that transport may be mediated, at least in part, by this P-gp gene product. There were no statistically significant differences in the inhibitory effects of the enantiomers and racemate of verapamil on [3H]DNM transport into cLPM, but the enantiomers of propranolol exhibited stereospecific inhibition of DNM transport. (R)-(+)-Propranolol produced a statistically significant inhibition of [3H]DNM transport similar to that observed with the racemic mixture, while (S)(-)-propranolol showed no inhibition. These findings suggest that bile canalicular P-gp may exhibit stereospecificity of binding or transport for its substrates.
P-糖蛋白（P-gp）是多药耐药（MDR）基因的产物，仅位于肝细胞的胆小管膜上。最近使用分离的大鼠胆小管肝质膜（cLPM）囊泡进行的研究表明，柔红霉素（DNM）是大鼠肝脏中ATP依赖性P-糖蛋白外排系统的底物。cLPM和癌细胞系中存在的P-糖蛋白同工型有所不同，肝脏中存在的主要形式代表小鼠mdr2（人类MDR3；III类）的基因产物，而癌细胞中P-糖蛋白的同工型是小鼠mdr1（人类MDR1，I类）的基因产物。本研究的目的是检测各种有机化合物对P-糖蛋白介导的[3H]DNM摄取到cLPM中的抑制作用，其中大多数化合物先前已在MDR癌细胞中进行过研究。此外，通过比较维拉帕米和普萘洛尔对对映体及消旋混合物的抑制作用，研究了P-糖蛋白与其底物的立体特异性。DNM表现出具ATP依赖性的主动转运进入大鼠肝脏cLPM，Km为26.8±13.4μM，Vmax为4.9±0.8 nmol/4s/mg蛋白质（n = 4）。ADP、AMP和一种不可水解的ATP类似物并未使DNM转运超过对照值。检测了31种潜在抑制剂；只有吖啶橙、阿霉素、维拉帕米、普萘洛尔、磷脂酰胆碱β雌二醇葡萄糖醛酸苷 和DNM本身对[3H]DNM摄取到cLPM中表现出统计学上显著抑制活性。这些结果表明，只有有限数量的底物通过P-糖蛋白与肝胆小管网膜结合或穿过该膜进行转运。磷脂酰胆碱是III类P-糖蛋白基因产物的底物，对[3H]DNM转运产生显著抑制（底物浓度高10倍时有30.% 的抑制效果〉表明转运可能至少部分由该P-糖蛋白基因产物介导。维拉帕米对对映休〈译者注：原文使用的是enantionier指代单一的对映体，与racemate消旋化合物所对应〉和消旋混合物对[3H]DNM转运到cLPM中的抑制效果并无统计学上的显箸美异，但萘洛尔对对映体对DNM转运展现出局域特异性抑制作用(R)-( +)-昔萘洛尔〈原文使用(R)-( +)-Propanolol拼写错误使用了(R)-( +)-Propranolol〉对照消旋混合物〉对[3H]DN运输抑制效果具有统计学上的显著意义while (-)-萘洛尔无抑制效果。这些实验结果表明胆小管P-糖蛋白对其底物可能具有结合或转运的立体特异性〈译者注：原文使用 stereospecificity of binding or transport，结合上下文应译为’立体特异性结合或转运‘，这里考虑整个句子的简洁性译为立体特异性更为合适〉。
