Effect of HEPES buffer on the uptake and transport of P-glycoprotein substrates and large neutral amino acids

Abstract

HEPES has been widely employed as an organic buffer agent in cell culture medium as well as uptake and transport experiments in vitro. However, concentrations of HEPES used in such studies vary from one laboratory to another. In this study, we investigated the effect of HEPES on the uptake and bidirectional transport of P-gp substrates employing both Caco-2 and MDCK-MDR1 cells. ATP-dependent uptake of glutamic acid was also examined. ATP production was further quantified applying ATP Determination Kit. An addition of HEPES to the growth and incubation media significantly altered the uptake and transport of P-gp substrates in both Caco-2 and MDCK-MDR1 cells. Uptake of P-gp substrates substantially diminished as the HEPES concentration was raised to 25 mM. Bidirectional (A-B and B-A) transport studies revealed that permeability ratio of P(appB-A) to P(appA-B) in the presence of 25 mM HEPES was significantly higher than control. The uptake of phenylalanine is an ATP-independent process, whereas the accumulation of glutamic acid is ATP-dependent. While phenylalanine uptake remained unchanged, glutamic acid uptake was elevated with the addition of HEPES. Verapamil is an inhibitor of P-gp mediated uptake; elevation of cyclosporine uptake in the presence of 5 muM verapamil was compromised by the presence of 25 mM HEPES. The results of ATP assay indicated that HEPES stimulated the production of ATP. This study suggests that the addition of HEPES in the medium modulated the energy dependent efflux and uptake processes. The effect of HEPES on P-gp mediated drug efflux and transport may provide some mechanistic insight into possible reasons for inconsistencies in the results reported from various laboratories.

Fig. 1

Uptake of [³H]Cyclosporine-A in DPBS buffer containing 0 mM, 15 mM, and 25 mM HEPES into different cell lines. A: Caco-2 cells; B: MDCK-MDR1 cells. Results were expressed as mean ± SD, n = 4 – 6. * p < 0.05; ** p < 0.01 (from control, 0 mM HEPES).

Fig. 2

Uptake of different P-gp substrates into MDCK-MDR1 cells in DPBS buffer containing 0 mM, 15 mM, and 25 mM HEPES. A: [³H]ritonavir; B: [³H]lopinavir. Results were expressed as mean ± SD, n = 4 – 8. * p < 0.05; ** p < 0.01 (from control, 0 mM HEPES).

Fig. 3

Cumulative amount of [³H]lopinavir transported with time (A-B and B-A) across MDCK-MDR1 cells in DPBS buffer containing 0 mM and 25 mM HEPES. Results were expressed as mean ± SD, n = 3 – 6.

Fig. 4

Uptake of different L- amino acids into MDCK-MDR1 cells in DPBS buffer containing 0 mM, 15 mM, and 25 mM HEPES. A: [³H]glutamic acid; B: [³H]phenylalanine. Results were expressed as mean ± SD, n = 4 – 6. * p < 0.05 (from control, 0 mM HEPES).

Fig. 5

Uptake of [³H]cyclosporine-A into Caco-2 cells in DPBS buffer containing 0 mM, 25 mM HEPES and with addition of 5 μM of verapamil, respectively. Results were expressed as mean ± SD, n = 4 – 6. * p < 0.05 (from control, 0 mM HEPES).

Fig. 6

Uptake of [³H]lopinavir in DPBS buffer containing 0 mM, 15 mM, and 25 mM HEPES into different sets of MDCK-MDR1 cells. Set 1: cells grown in culture medium without HEPES; Set 2: cells grown in culture medium with addition of 20 mM HEPES. Results were expressed as mean ± SD, n = 4 – 6. * p < 0.05; ** p < 0.01 (Set 2 compared with Set 1).

Fig. 7

Uptake of [³H]lopinavir in the presence of different concentrations of unlabelled lopinavir (1 μM, 2 μM and 5 μM) into different sets of MDCK-MDR1 cells. Set 1: cells grown in culture medium without HEPES; Set 2: cells grown in culture medium with addition of 20 mM HEPES. Results were expressed as mean ± SD, n = 4 – 6. ** p < 0.01 (Set 2 compared with Set 1).

Fig. 8

Effect of HEPES on ATP production in MDCK-MDR1 cells. Set 1: cells grown in culture medium without HEPES; Set 2: cells grown in culture medium with addition of 20 mM HEPES. Results were expressed as mean ± SD, n = 4 – 6. ** p < 0.01 (Set 2 compared with Set 1, Set 1 as control).