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Unstirred Water Layers and the Kinetics of Organic Cation Transport

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Abstract

Purpose

Unstirred water layers (UWLs) present an unavoidable complication to the measurement of transport kinetics in cultured cells, and the high rates of transport achieved by overexpressing heterologous transporters exacerbate the UWL effect. This study examined the correlation between measured Jmax and Kt values and the effect of manipulating UWL thickness or transport Jmax on the accuracy of experimentally determined kinetics of the multidrug transporters, OCT2 and MATE1.

Methods

Transport of TEA and MPP was measured in CHO cells that stably expressed human OCT2 or MATE1. UWL thickness was manipulated by vigorous reciprocal shaking. Several methods were used to manipulate maximal transport rates.

Results

Vigorous stirring stimulated uptake of OCT2-mediated transport by decreasing apparent Kt (Ktapp) values. Systematic reduction in transport rates was correlated with reduction in Ktapp values. The slope of these relationships indicated a 1500 μm UWL in multiwell plates. Reducing the influence of UWLs (by decreasing either their thickness or the Jmax of substrate transport) reduced Ktapp by 2-fold to >10-fold.

Conclusions

Failure to take into account the presence of UWLs in experiments using cultured cells to measure transport kinetics can result in significant underestimates of the apparent affinity of multidrug transporters for substrates.

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Abbreviations

CHO:

Chinese hamster ovary

DDI:

Drug-drug interaction

HEK-293:

Human embryonic kidney 293

hMATE1:

human Multidrug And Toxin Extruder 1

hOCT2:

human Organic Cation Transporter 2

Jmax :

Maximal rate of transport

Kt :

Michaelis constant

Ktapp :

Apparent Michaelis constant

MDCK:

Madin-Darby canine kidney

MPP:

1-Methyl-4-phenylpyridinium

NBD-MTMA:

N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][l,2,5]oxadiazol-4-yl)amino]ethanaminium

TEA:

Tetraethylammonium

UWL:

Unstirred water layer

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ACKNOWLEDGEMENTS AND DISCLOSURES

This work was supported in part by NIH grants 5R01DK058251, 1R01DK080801, 5P30ES006694, and 5T32HL07249. The authors extend their thanks to Dr. William H. Dantzler, University of Arizona, and Dr. Ryan M. Pelis, Dalhousie University, for helpful discussions.

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Author notes

  1. Takahiro Shibayama

    Present address: Translational Research, TMCP, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo, 140-8710, Japan

Authors and Affiliations

  1. Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona, 85724, USA

    Takahiro Shibayama, Mark Morales, Xiaohong Zhang, Lucy J. Martínez-Guerrero, Timothy W. Secomb & Stephen H. Wright

  2. GÉPROM (Groupe d’Études des Protéines Membranaires) Département de Physiologie, Université de Montréal, Montréal, Quebec, H3C 3J7, Canada

    Alfred Berteloot

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  1. Takahiro Shibayama
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  3. Xiaohong Zhang
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Correspondence to Stephen H. Wright.

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Shibayama, T., Morales, M., Zhang, X. et al. Unstirred Water Layers and the Kinetics of Organic Cation Transport. Pharm Res 32, 2937–2949 (2015). https://doi.org/10.1007/s11095-015-1675-5

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