Age-dependent expression of P-glycoprotein gp170 in Caco-2 cell monolayers

Pharm Res. 1996 Jun;13(6):885-90. doi: 10.1023/a:1016005212640.

Abstract

Purpose: 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / biosynthesis*
  • Biological Transport, Active
  • Blotting, Western
  • Caco-2 Cells
  • Calcium Channel Blockers / pharmacology
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism
  • Cyclosporine / metabolism
  • Dexamethasone / metabolism
  • Diuretics, Osmotic / metabolism
  • Glucocorticoids / metabolism
  • Humans
  • Immunosuppressive Agents / metabolism
  • Mannitol / metabolism
  • Permeability
  • Time Factors
  • Verapamil / pharmacology

Substances

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Calcium Channel Blockers
  • Diuretics, Osmotic
  • Glucocorticoids
  • Immunosuppressive Agents
  • Mannitol
  • Dexamethasone
  • Cyclosporine
  • Verapamil