A new procedure for the quantitative assessment of p-glycoprotein efflux pump associated with human T lymphocytes.
BACKGROUND
P-glycoprotein (P-gp), a multidrug transporter located in plasma membranes, reduces intracellular availability of some drugs. Upregulation of P-gp has been observed in some clinical situations, including chronic inflammatory disease and viral infection. However, P-gp is expressed in only a small subset of peripheral blood mononuclear cells (PBMC) and at much lower quantities than it is on P-gp-positive cell lines used in other studies.


METHODS
P-gp expression was assessed by flow cytometry by using a commercially available, anti-P-gp, allophycocyanin-conjugated monoclonal antibody. Flow cytometry was also used to determine the efflux activity associated with P-gp; with this process, refluxed fluorescent P-gp substrate, rhodamine 123 (Rho123), was determined by the subsequently identified P-gp-positive PBMC subset. Use of verapamil during the dye-loading procedure maximized the amount of dye retained by the cells.


RESULTS
The use of allophycocyanin-conjugated monoclonal antibody allowed for the identification of P-gp-positive PBMC subsets, even when the cells were fully loaded with Rho123. We used a logical gating strategy to identify a P-gp-positive PBMC subset, after which P-gp efflux activity of the PBMC subset could be quantitatively assessed. This new procedure enabled us to assess the P-gp efflux function of T lymphocytes in some clinical situations, which induced P-gp upregulation in vivo.


CONCLUSIONS
This new procedure enables us to quantitatively assess the P-gp efflux activity associated with PBMC.
Vélez
Rosa
R
AIDS Research Program, Ponce School of Medicine, Ponce, Puerto Rico, USA. rosaamely@yahoo.com
Sánchez
María del C
Mdel C
Lopez
Pablo
P
Yamamura
Yasuhiro
Y
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis*; D000911:Antibodies, Monoclonal; D002121:Calcium Channel Blockers / Q000494:pharmacology; D019970:Cocaine-Related Disorders / Q000175:diagnosis; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D020112:Rhodamine 123; D013601:T-Lymphocytes / Q000378:metabolism*; D015854:Up-Regulation; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D002121:Calcium Channel Blockers; D005456:Fluorescent Dyes; D020112:Rhodamine 123; D014700:Verapamil