DMCA
Effect of HEPES buffer on the uptake and transport of P- glycoprotein substrates and large neutral amino acids
BibTeX
@MISC{Luo_effectof,
author = {Shuanghui Luo and Dhananjay Pal and Sujay J. Shah and Deep Kwatra and Kalyani D. Paturi and K. Mitra},
title = {Effect of HEPES buffer on the uptake and transport of P- glycoprotein substrates and large neutral amino acids},
year = {}
}
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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 cellular washing 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 PappB-A to PappA-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 μM 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.
Keyphrases
large neutral amino acid p-gp substrate glycoprotein substrate hepes buffer mdck-mdr1 cell mm hepes glutamic acid uptake process possible reason incubation medium atp-independent process various laboratory transport study cyclosporine uptake hepes concentration energy dependent efflux organic buffer agent drug efflux phenylalanine uptake atp determination kit bidirectional transport transport experiment cellular washing unchanged glutamic acid uptake atp-dependent uptake cell culture medium atp production mechanistic insight permeability ratio
