Abstract
Gp170 (also known as P-glycoprotein) is a transmembrane glycoprotein which is overexpressed in multidrug-resistant tumor cells and is also found in the apical plasma membrane domain of several normal human and animal tissues. Gp170 has been postulated to function as an energy-dependent efflux pump for cytotoxic drugs. In rat liver, Gp170 is restricted to the bile canalicular domain of the plasma membrane. Canalicular membrane vesicles (CMV), but not sinusoidal membrane vesicles, contained a approximately 160-kDa protein which reacts with anti-Gp170 monoclonal antibody and manifest ATP-dependent [3H]daunomycin transport which is temperature dependent, osmotically sensitive, and saturable. Among several nucleotides, ATP was a potent stimulator of transport whereas non- or slowly hydrolyzable analogues (adenosin-5-O-(3-thiotriphosphate, adenyl-5-yl-imidodiphosphate) were ineffective. ATP-dependent daunomycin transport was inhibited by cytotoxic drugs (vinblastine, vincristine, and adriamycin) and other drugs, such as verapamil and quinidine, which restore anti-cancer drug sensitivity in resistant cells. Inside-out CMV were separated from right side-out CMV by antibody-induced affinity density perturbation. Only inside-out CMV manifested ATP-dependent daunomycin transport. These results suggest that Gp170 is an ATP-dependent efflux pump which is responsible for the undirectional, energy-dependent transport of daunomycin and other drugs by rat liver into the bile.
Highlights
Among several nucleotides, ATP was a potent stim- branes and present evidence that Gp170 is a transporter of ulator of transport whereas non- osrlowly hydrolyza- cytotoxic and other drugs
Several investigators identified a gene which is associated with the multidrug-resistant phenotype [6,7,8,9,10,11] and a membrane glycoprotein (Gp170) which is encoded by the Preparation and Characterization of Vesicles-Canalicular membrane vesicles (CMV) and sinusoidal membrane vesicles (SMV) were separately prepared from male Sprague-Dawley rats weighing 200300g.CMV were isolated from liver homogenates using nitrogen gene has been identified [12, 13]
10%of daunomycin associated with vesicles in theabsence of ATP responded to increased medium osmolality. These results indicate that daunomycin is transported into CMV in mM unlabeled ATP and quenching with trichloroacetate
Summary
Cin) and other drugs, sucahs verapamil and quinidine, which restore anti-cancer drusgensitivity in resistant. 10%of daunomycin associated with vesicles in theabsence of ATP responded to increased medium osmolality These results indicate that daunomycin is transported into CMV in mM unlabeled ATP and quenching with trichloroacetate. Samples transport by CMV was determined after 1min of incubation were washed with 0.15 M potassium phosphate, pH 2, dissolved in solution containing 5% 2-mercaptoethanol, 0.25 M sucrose, 0.1 M potassiumphosphate, pH 4, and 70 mM hexadecylpyridiniumchloride, and applied to acid gel electrophoresis as described previously [31]. Blots were blocked with Tris-buffered saline containing 3% bovine serum albumin and sequentially incubated with a monoclonal anti-Gpl7O antibody (C219, 1 pg/ml) for 12-18 h a t 4 "C, and proteinA horseradish peroxidase conjugate measured as a function of ATP concentration (Fig. 4). Antigen was detected by using mycin transport in theabsence of an ATP-regenerating syshorseradish peroxidase color development (Bio-Rad)
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