Assessment of P-glycoprotein-dependent drug transport in isolated rat hepatocytes using rhodamine 123.
To test the activity of P-170 glycoprotein in isolated hepatocytes, a method has been developed employing the fluorescent dye rhodamine 123 (R-123). The uptake of R-123 by both freshly isolated and 4-hr-plated hepatocytes depends on dye concentration, time of incubation, and cell number. The efflux of R-123 from cells is inhibited by sodium azide and by verapamil. In standard conditions the efficiency of efflux of R-123 from cells correlates with the relative amount of immunoblottable glycoprotein. The method has been applied to detection of P-170 activity in hepatocytes from animals of different ages as well as from carcinogen-treated animals. The proposed assay appears a simple and adequate tool for the functional assessment of multidrug transporter in liver.
Chieli
E
E
Dipartimento de Biomedicina Sperimentale, Università di Pisa, Italy.
Santoni Rugiu
E
E
Cervelli
F
F
Paolicchi
A
A
Tongiani
R
R
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D001386:Azides / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004351:Drug Resistance; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020112:Rhodamine 123; D012235:Rhodamines / Q000493:pharmacokinetics; D019810:Sodium Azide; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D012235:Rhodamines; D020112:Rhodamine 123; D019810:Sodium Azide; D014700:Verapamil