The expression in intestinal epithelia of transport proteins such as the multidrug resistance-associated proteins (MRPs) may limit the bioavailablity of many drugs.
We have previously shown when Caco-2 cells are loaded with calcein (an MRP substrate; Hollo et al. 1996), efflux is by a probenecid-sensitive mechanism (Prime et al. 1999). This efflux is approximately 2-fold greater to the apical solution than to the basal (5.4 ± 0.3 and 2.3 ± 0.2 % efflux (mean ± S.E.M. n = 12), respectively). In the presence of sodium azide and 2-deoxyglucose (to deplete ATP), apical efflux was reduced to 12.5 ± 0.6 % of control; basal efflux was 46.3 ± 13.6 % of control (n = 12). The MRP inhibitor MK571 (10 µM) also reduced efflux (37.0 ± 4.7 and 65.6 ± 7.0 % of control for apical and basal efflux, respectively, n = 12). These results suggest MRP involvement in both apical and basal efflux.
To assess which MRP isoforms may be involved in calcein efflux, specific primers to each of the six MRPs (Kool et al. 1997) were designed. Reverse-transcription polymerase chain reactions (RT-PCR) were performed and products were separated on agarose gels. Products of the expected size were cloned and sequenced from all six primer sets. The products were shown to have > 99 % homology to the respective MRP. Using optimal PCR conditions, multiplex PCR was performed to amplify the MRP of interest along with 18S rRNA as an invariant endogenous control. MRP2 was the predominant isoform and shown to be localised to apical membranes of Caco-2 cells by immunostaining.
We have demonstrated all six MRP isoforms to be expressed in Caco-2 cells, extending the studies of Hirohashi et al. (2000). We have also shown that functional MRP activity is present at both apical and basal membranes of Caco-2 cells. MRP2 has the correct localisation and dominance to be involved in apical efflux.
This work was supported by AstraZeneca.
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