Unstirred Water Layers and the Kinetics of Organic Cation Transport

Takahiro Shibayama; Mark Morales; Xiaohong Zhang; Lucy J. Martínez-Guerrero; Alfred Berteloot; Timothy W. Secomb; Stephen H. Wright

doi:10.1007/s11095-015-1675-5

Unstirred Water Layers and the Kinetics of Organic Cation Transport

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

Research output: Contribution to journal › Article › peer-review

20 Scopus citations

Abstract

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 J_max and K_t values and the effect of manipulating UWL thickness or transport J_max 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 K_t (K_tapp) values. Systematic reduction in transport rates was correlated with reduction in K_tapp values. The slope of these relationships indicated a 1500Â

Original language	English (US)
Article number	1675
Pages (from-to)	2937-2949
Number of pages	13
Journal	Pharmaceutical Research
Volume	32
Issue number	9
DOIs	https://doi.org/10.1007/s11095-015-1675-5
State	Published - Sep 8 2015

Keywords

kinetics
organic cation
transport
unstirred water layers

ASJC Scopus subject areas

Biotechnology
Molecular Medicine
Pharmacology
Pharmaceutical Science
Organic Chemistry
Pharmacology (medical)

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10.1007/s11095-015-1675-5

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title = "Unstirred Water Layers and the Kinetics of Organic Cation Transport",

abstract = "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{\^A}",

keywords = "kinetics, organic cation, transport, unstirred water layers",

author = "Takahiro Shibayama and Mark Morales and Xiaohong Zhang and Mart{\'i}nez-Guerrero, {Lucy J.} and Alfred Berteloot and Secomb, {Timothy W.} and Wright, {Stephen H.}",

note = "Funding Information: 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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doi = "10.1007/s11095-015-1675-5",

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AU - Shibayama, Takahiro

AU - Morales, Mark

AU - Zhang, Xiaohong

AU - Martínez-Guerrero, Lucy J.

AU - Berteloot, Alfred

AU - Secomb, Timothy W.

AU - Wright, Stephen H.

N1 - Funding Information: 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.

PY - 2015/9/8

Y1 - 2015/9/8

N2 - 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 1500Â

AB - 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 1500Â

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Unstirred Water Layers and the Kinetics of Organic Cation Transport

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