Phorbol 12-myristate 13-acetate inhibits P-glycoprotein-mediated efflux of digoxin in MDCKII-MDR1 and Caco-2 cell monolayer models.

Aim: To investigate the effects of phorbol 12-myristate 13-acetate (PMA), a PKC activator, on P-glycoprotein-mediated efflux of digoxin in two cell transport models.

Methods: Caco-2 cells, wild MDCKII cells (MDCKII-WT) and MDCKII cells transfected stably with human MDR1-gene encoding P-gp (MDCKII-MDR1) were examined. Cell viability was evaluated with MTT assay. Bidirectional transport of digoxin was evaluated in these cells. Intracellular ATP level was measured using ATP assay. P-gp ATPase activity was analyzed using a Pgp-Glo(TM) assay.

Results: PMA (10 μmol/L) did not reduce the viability of the 3 types of cells. In Caco-2 and MDCKII-MDR1 cell monolayers, PMA (1, 10 and 100 nmol/L) dose-dependently inhibited the basolateral to apical transport of digoxin, but did not change the apical to basolateral transport. In addition, PMA did not affect both the basolateral to apical and apical to basolateral transport of digoxin in MDCKII-WT cell monolayer. In agreement with the above results, PMA dose-dependently reduced intracellular ATP level and stimulated P-gp ATPase activity in both Caco-2 and MDCKII-MDR1 cells. Verapamil (a positive control, 100 μmol/L) caused similar inhibition on digoxin efflux as PMA did, whereas 4α-PMA (a negative control, 100 nmol/L) had no effect.

Conclusion: PMA significantly inhibited P-gp-mediated efflux of digoxin in both Caco-2 and MDCKII-MDR1 cell monolayers via PKC activation.