Document Detail


Effect of HEPES buffer on the uptake and transport of P-glycoprotein substrates and large neutral amino acids.
MedLine Citation:
PMID:  20163160     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Shuanghui Luo; Dhananjay Pal; Sujay J Shah; Deep Kwatra; Kalyani D Paturi; Ashim K Mitra
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural    
Journal Detail:
Title:  Molecular pharmaceutics     Volume:  7     ISSN:  1543-8392     ISO Abbreviation:  Mol. Pharm.     Publication Date:  2010 Apr 
Date Detail:
Created Date:  2010-04-05     Completed Date:  2010-06-29     Revised Date:  2013-05-30    
Medline Journal Info:
Nlm Unique ID:  101197791     Medline TA:  Mol Pharm     Country:  United States    
Other Details:
Languages:  eng     Pagination:  412-20     Citation Subset:  IM    
Affiliation:
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri 64108-2718, USA.
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MeSH Terms
Descriptor/Qualifier:
Adenosine Triphosphate / metabolism
Amino Acids, Neutral / metabolism*
Animals
Biological Transport / drug effects*
Caco-2 Cells
Cell Line
Cell Membrane Permeability / drug effects
Cyclosporine / metabolism
Dogs
Glutamic Acid / metabolism
HEPES / pharmacology*
Humans
Lopinavir
P-Glycoprotein / metabolism*
Phenylalanine / metabolism
Pyrimidinones / metabolism
Ritonavir / metabolism
Verapamil / pharmacology
Grant Support
ID/Acronym/Agency:
R01 A1 071199//PHS HHS; R01 AI071199-01A2/AI/NIAID NIH HHS; R01 AI071199-02/AI/NIAID NIH HHS; R01 AI071199-03/AI/NIAID NIH HHS; R01 GM 64320/GM/NIGMS NIH HHS; R01 GM064320-01A2/GM/NIGMS NIH HHS; R01 GM064320-02/GM/NIGMS NIH HHS; R01 GM064320-03/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Amino Acids, Neutral; 0/Lopinavir; 0/P-Glycoprotein; 0/Pyrimidinones; 0/Ritonavir; 52-53-9/Verapamil; 56-65-5/Adenosine Triphosphate; 56-86-0/Glutamic Acid; 59865-13-3/Cyclosporine; 63-91-2/Phenylalanine; 7365-45-9/HEPES
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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