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. 1998 Mar;87(3):300-5. doi: 10.1021/js970290q.
P-glycoprotein (P-gp), the multidrug resistance gene product, is expressed in a normal liver exclusively on the canalicular membrane of the hepatocyte. The objective of this study was to examine the effect of age on the P-gp transport system using canalicular membrane (cLPM) vesicles isolated from the liver of developing (22 days old) and adult rats. No differences in protein yield, intravesicular volumes, and enrichments of cLPM enzymes or enzymes representing contamination of subcellular organelles were found for vesicles isolated from both groups, demonstrating the isolation of similar cLPM vesicle preparations. The transport of daunomycin (DNM), a P-gp substrate, was used to study age-related functional differences in P-gp. DNM uptake in the presence of ATP was greater than uptake in the absence of ATP in both young and adult cLPM vesicles, showing that P-gp is functional in both groups. In young and adult groups only ATP was a potent stimulator of transport when compared with ATP degradation products and a nonhydrolyzable ATP analogue. Although ATP-dependent uptake tended to be greater in the adult compared to the young, there was no statistically significant difference in DNM kinetics (Vmax, km, gamma) between groups. Canalicular membrane from the young rats showed decreased fluidity, as assessed by the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene; however there was no significant difference between groups. Examination of P-gp expression using the monoclonal antibody C219 revealed similar levels of expression in the young as in the adult. Our results suggest that P-gp in the bile canaliculus of developing rats is functional with similar levels of function and expression as observed in the adult.
多药耐药基因产物P-糖蛋白（P-gp）在正常肝脏中仅表达于肝细胞的胆小管膜上。本研究的目的是使用从发育中（22日龄）和成年大鼠肝脏分离的胆小管膜（cLPM）囊泡，研究年龄对P-gp转运系统的影响。从两组分离的囊泡在蛋白质产量、囊泡内体积以及cLPM酶或代表亚细胞器污染的酶的富集方面均未发现差异，这表明分离得到了相似的cLPM囊泡制剂。使用P-gp底物柔红霉素（DNM）的转运来研究P-gp与年龄相关的功能差异。在有ATP存在的情况下，幼年和成年cLPM囊泡对DNM的摄取均大于无ATP时的摄取，表明两组中的P-gp均具有功能。与ATP降解产物和不可水解的ATP类似物相比，在幼年和成年组中只有ATP是转运的有效刺激剂。尽管成年组中ATP依赖性摄取往往比幼年组更大，但两组之间DNM动力学（Vmax、km、γ）没有统计学上的显著差异。通过1,6-二苯基-1,3,5-己三烯的荧光偏振评估，幼年大鼠的胆小管膜流动性降低；然而，两组之间没有显著差异。使用单克隆抗体C219检测P-gp表达，结果显示幼年和成年组的表达水平相似。我们的结果表明，发育中大鼠胆小管中的P-gp具有功能，其功能和表达水平与成年大鼠相似。
