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Functional expression of P-glycoprotein in the hepatic canalicular membrane of developing rats
1Department of Pharmaceutics, School of Pharmacy, State University of New York at Buffalo, Amherst 14260, USA.
Insights
P-glycoprotein (P-gp) function and expression in rat liver canalicular membrane vesicles are similar in developing and adult animals. This suggests P-gp is fully functional early in life, aiding in drug transport.
Area of Science:
- Hepatology
- Pharmacology
- Cell Biology
Background:
- P-glycoprotein (P-gp) is crucial for multidrug resistance and expressed on hepatocyte canalicular membranes.
- Understanding age-related P-gp function is vital for pediatric drug development and pharmacokinetics.
Purpose of the Study:
- To investigate the impact of age on the P-gp transport system in rat liver canalicular membrane vesicles.
- To compare P-gp function, expression, and kinetics between developing and adult rats.
Main Methods:
- Isolation of canalicular membrane (cLPM) vesicles from developing (22-day-old) and adult rat livers.
- Assessing P-gp function via daunomycin (DNM) uptake assays in the presence/absence of ATP.
- Evaluating membrane fluidity and P-gp expression using monoclonal antibody C219.
Main Results:
- Vesicle preparations were comparable in protein yield, volume, and enzyme enrichment between age groups.
- P-gp demonstrated ATP-dependent daunomycin transport in both young and adult rats.
- No significant differences in daunomycin kinetics (Vmax, km, gamma) or P-gp expression were observed between developing and adult rats.
Conclusions:
- P-glycoprotein in the bile canaliculus of developing rats is functional.
- P-gp exhibits similar functional levels and expression in developing and adult rats, indicating early maturation of this transporter system.
Abstract:
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.