Demonstration of the activity of P-glycoprotein by a semi-automated fluorometric method.
BACKGROUND
We have developed a semi-automated fluorometric method that utilizes ethidium bromide (EB), a common substrate of bacterial efflux pumps. The method is sufficiently sensitive to characterize the efflux pump systems of bacteria. Because EB is also recognized and extruded by ATP-binding cassette (ABC) transporters and these have similarity to P-glycoprotein (P-gp), the method has been extended for the evaluation of agents that can inhibit the extrusion of EB on a real-time basis by mouse lymphoma cells containing the human ABCB1 (mdr1) gene.


METHODS
Monitoring of uptake and extrusion of EB was assessed using the Rotor-Gene 3000 (Corbett Research) under different conditions.


RESULTS
Whereas extrusion of EB took place readily, the addition of known inhibitors of efflux pumps (verapamil, reserpine) caused retention of EB.


CONCLUSIONS
This method is inexpensive and allows the detection of neoplastic cells with increased efflux activity as well as the screening of large numbers of compounds for inhibition of the P-gp.
Spengler
Gabriella
G
Unit of Mycobacteriology, UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), 1349-008 Lisboa, Portugal.
Viveiros
Miguel
M
Martins
Marta
M
Rodrigues
Liliana
L
Martins
Ana
A
Molnar
Jozsef
J
Couto
Isabel
I
Amaral
Leonard
L
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D018759:Adrenergic Uptake Inhibitors / Q000494:pharmacology; D000818:Animals; D001331:Automation; D001692:Biological Transport / Q000187:drug effects; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D004996:Ethidium / Q000378:metabolism*; D005470:Fluorometry; D006801:Humans; D016399:Lymphoma, T-Cell / Q000378:metabolism*; D051379:Mice; D012110:Reserpine / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018759:Adrenergic Uptake Inhibitors; D002121:Calcium Channel Blockers; D012110:Reserpine; D014700:Verapamil; D004996:Ethidium