Age-dependent expression of P-glycoprotein gp170 in Caco-2 cell monolayers.
OBJECTIVE
To determine whether the expression and activity of the P-glycoprotein (P-GP) drug efflux pump vary with the culture age of Caco-2 cell monolayers.


METHODS
Caco-2 cell monolayers were grown for 3-27 days on tissue culture-treated Transwells. P-GP efflux function was determined by measuring transmonolayer fluxes of cyclosporin A (CsA) and verapamil, while P-GP expression level was evaluated by Western blot analysis using monoclonal antibody C219.


RESULTS
The apparent permeability coefficient (Papp) of CsA (0.5 microM) in the basolateral-to-apical (B-->A) direction increased with culture age and was higher than the apical-to-basolateral (A-->B) direction at all times. Net secretory Papp significantly increased from day 17 onward compared to that observed during day 3 through 13. Verapamil (100 microM) significantly inhibited CsA transport in the B-->A direction from day 17 to 27, while elevating CsA transport in the A-->B direction from day 6 to 27. Interestingly, the Papp of verapamil (0.5 microM) in the B-->A direction was significantly higher than in the A-->B direction from day 6 to 27, rendering increases in net secretory Papp of verapamil with culture age. Western analysis revealed that P-GP expression level was in the order of 4 weeks approximately 1 week > 3 weeks > 2 weeks at equal loading of cell proteins.


CONCLUSIONS
P-GP is continuously expressed throughout the culture period, but it may not be fully functional at an early age. Caco-2 cell monolayers of day 17 to 27 appear to be a good model to evaluate the functional role of P-GP in drug efflux.
Hosoya
K I
KI
University of Southern California, School of Pharmacy, Department of Pharmaceutical Sciences, Los Angeles 90033, USA.
Kim
K J
KJ
Lee
V H
VH
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis*; D001693:Biological Transport, Active; D015153:Blotting, Western; D018938:Caco-2 Cells; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D016572:Cyclosporine / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism; D004234:Diuretics, Osmotic / Q000378:metabolism; D005938:Glucocorticoids / Q000378:metabolism; D006801:Humans; D007166:Immunosuppressive Agents / Q000378:metabolism; D008353:Mannitol / Q000378:metabolism; D010539:Permeability; D013997:Time Factors; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002121:Calcium Channel Blockers; D004234:Diuretics, Osmotic; D005938:Glucocorticoids; D007166:Immunosuppressive Agents; D008353:Mannitol; D003907:Dexamethasone; D016572:Cyclosporine; D014700:Verapamil