Kinetic analysis of P-glycoprotein-mediated transport by using normal human placental brush-border membrane vesicles.
OBJECTIVE
P-Glycoprotein (Pgp) plays an important role in drug disposition and excretion in various tissues such as the brain, intestine, and kidney. Moreover, we have demonstrated that Pgp is expressed on the brush-border membranes of trophoblast cells in the placenta and restricts drug transfer from the maternal circulation to the fetus. However, the transport kinetics of physiologically expressed Pgp has scarcely been investigated.


METHODS
In this study, we assessed the functional kinetics of transport mediated by Pgp that is physiologically expressed in normal tissue by using human placental brush-border membrane vesicles (BBMVs). Digoxin and vinblastine were used as typical substrates of Pgp.


RESULTS
The uptakes of [3H]digoxin and [3H]vinblastine into BBMVs were significantly increased in the presence of an ATP-regenerating system. The ATP-dependent uptakes of [3H]digoxin and [3H]vinblastine into BBMVs exhibited saturable kinetics. The Michaelis constants (Kt values) were 2.65 +/- 1.80 microM and 21.9 +/- 3.37 microM, respectively. In the presence of a Pgp inhibitor such as verapamil, cyclosporine A, or progesterone, the ATP-dependent uptakes of [3H]digoxin and [3H]vinblastine into BBMVs were significantly reduced. Anti-Pgp monoclonal antibody C219 completely inhibited the uptake of [3H]digoxin.


CONCLUSIONS
The transport kinetics of [3H]digoxin and [3H]vinblastine by physiologically expressed Pgp were successfully evaluated by using BBMVs prepared from normal human placenta. The present method enabled us to evaluate the function of physiologically expressed Pgp and is superior to the use of cultured transfectants in terms of the yield of vesicles. The present method may also be applicable to investigating the influence of various factors such as the genotype of the MDR1 gene or various pathophysiologic states of neonates on the function of Pgp.
Ushigome
Fumihiko
F
Department of Medico-Pharmaceutical Sciences, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Koyabu
Noriko
N
Satoh
Shoji
S
Tsukimori
Kiyomi
K
Nakano
Hitoo
H
Nakamura
Takanori
T
Uchiumi
Takeshi
T
Kuwano
Michihiko
M
Ohtani
Hisakazu
H
Sawada
Yasufumi
Y
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000493:pharmacokinetics*; D001692:Biological Transport / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D005260:Female; D006801:Humans; D008871:Microvilli / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1