Quantification of biliary excretion and sinusoidal excretion of 5(6)-carboxy-2 ',7 '-dichlorofluorescein (CDF) in cultured hepatocytes isolated from Sprague Dawley, Wistar and Mrp2-deficient Wistar (TR-) rats

L. C. J. Ellis*, M. H. Grant, Gabrielle Margaret Hawksworth, R. J. Weaver

*Corresponding author for this work

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Hepatic efflux of drug candidates is an important issue in pre-clinical drug development. Here we utilise a method which quantifies and distinguishes efflux of drugs at the canalicular and sinusoidal membranes in rat hepatocyte cultures. Bi-phasic kinetics of transport of 5(6)-carboxydichlorofluorescein (CDF) at the canalicular membrane was demonstrated in Sprague Dawley (SD) and Wistar (W) rat hepatocytes. The high affinity component (Km = 3.2 +/- 0.8 mu M (SD), 9.0 +/- 3.1 mu M (W)) was attributed to Mrp2-mediated transport, the low affinity component (Km = 192.1 +/- 291.5 mu M (SD), 69.2 +/- 36.2 mu M (W)) may be attributed to transport involving a separate Mrp2 binding site. Data from membranes (Hill coefficient (h) = 2.0 +/- 0.5) and vesicles (h = 1.6 +/- 0.2) expressing Mrp2 and from SD (h = 1.6 +/- 0.4) and Wistar (h = 4.0 +/- 0.6) hepatocytes suggests transport involves more than one binding site. In TR- hepatocytes, CDF efflux was predominantly over the sinusoidal membrane (Km = 100.7 +/- 36.0 mu M), consistent with low abcc2 (Mrp2) expression and compensatory increase in abcc3 (Mrp3) expression. This report shows the potential of using this in vitro method to model changes in biliary excretion due to alterations in transporter expression. (C) 2014 Elsevier Ltd. All rights reserved.

Original languageEnglish
Pages (from-to)1165-1175
Number of pages11
JournalToxicology in Vitro
Volume28
Issue number6
Early online date4 Jun 2014
DOIs
Publication statusPublished - Sep 2014

Keywords

  • primary rat hepatocytes
  • membranes and vesicles expressing rat
  • Mrp2
  • drug transporters
  • canalicular efflux
  • sinusoidal efflux
  • Dubin-Johnson syndrome
  • collagen-sandwich configuration
  • canalicular networks
  • transport protein
  • expression
  • drug
  • MRP2
  • membrane
  • liver
  • disposition

Cite this

Quantification of biliary excretion and sinusoidal excretion of 5(6)-carboxy-2 ',7 '-dichlorofluorescein (CDF) in cultured hepatocytes isolated from Sprague Dawley, Wistar and Mrp2-deficient Wistar (TR-) rats. / Ellis, L. C. J.; Grant, M. H.; Hawksworth, Gabrielle Margaret; Weaver, R. J.

In: Toxicology in Vitro, Vol. 28, No. 6, 09.2014, p. 1165-1175.

Research output: Contribution to journalArticle

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title = "Quantification of biliary excretion and sinusoidal excretion of 5(6)-carboxy-2 ',7 '-dichlorofluorescein (CDF) in cultured hepatocytes isolated from Sprague Dawley, Wistar and Mrp2-deficient Wistar (TR-) rats",
abstract = "Hepatic efflux of drug candidates is an important issue in pre-clinical drug development. Here we utilise a method which quantifies and distinguishes efflux of drugs at the canalicular and sinusoidal membranes in rat hepatocyte cultures. Bi-phasic kinetics of transport of 5(6)-carboxydichlorofluorescein (CDF) at the canalicular membrane was demonstrated in Sprague Dawley (SD) and Wistar (W) rat hepatocytes. The high affinity component (Km = 3.2 +/- 0.8 mu M (SD), 9.0 +/- 3.1 mu M (W)) was attributed to Mrp2-mediated transport, the low affinity component (Km = 192.1 +/- 291.5 mu M (SD), 69.2 +/- 36.2 mu M (W)) may be attributed to transport involving a separate Mrp2 binding site. Data from membranes (Hill coefficient (h) = 2.0 +/- 0.5) and vesicles (h = 1.6 +/- 0.2) expressing Mrp2 and from SD (h = 1.6 +/- 0.4) and Wistar (h = 4.0 +/- 0.6) hepatocytes suggests transport involves more than one binding site. In TR- hepatocytes, CDF efflux was predominantly over the sinusoidal membrane (Km = 100.7 +/- 36.0 mu M), consistent with low abcc2 (Mrp2) expression and compensatory increase in abcc3 (Mrp3) expression. This report shows the potential of using this in vitro method to model changes in biliary excretion due to alterations in transporter expression. (C) 2014 Elsevier Ltd. All rights reserved.",
keywords = "primary rat hepatocytes, membranes and vesicles expressing rat, Mrp2, drug transporters, canalicular efflux, sinusoidal efflux, Dubin-Johnson syndrome, collagen-sandwich configuration, canalicular networks, transport protein, expression, drug, MRP2, membrane, liver, disposition",
author = "Ellis, {L. C. J.} and Grant, {M. H.} and Hawksworth, {Gabrielle Margaret} and Weaver, {R. J.}",
note = "Article Accepted Date: 21 May 2014 Acknowledgement: The authors would like to acknowledge Biologie Servier, for providing financial support for this work as a PhD studentship awarded to LE.",
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T1 - Quantification of biliary excretion and sinusoidal excretion of 5(6)-carboxy-2 ',7 '-dichlorofluorescein (CDF) in cultured hepatocytes isolated from Sprague Dawley, Wistar and Mrp2-deficient Wistar (TR-) rats

AU - Ellis, L. C. J.

AU - Grant, M. H.

AU - Hawksworth, Gabrielle Margaret

AU - Weaver, R. J.

N1 - Article Accepted Date: 21 May 2014 Acknowledgement: The authors would like to acknowledge Biologie Servier, for providing financial support for this work as a PhD studentship awarded to LE.

PY - 2014/9

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N2 - Hepatic efflux of drug candidates is an important issue in pre-clinical drug development. Here we utilise a method which quantifies and distinguishes efflux of drugs at the canalicular and sinusoidal membranes in rat hepatocyte cultures. Bi-phasic kinetics of transport of 5(6)-carboxydichlorofluorescein (CDF) at the canalicular membrane was demonstrated in Sprague Dawley (SD) and Wistar (W) rat hepatocytes. The high affinity component (Km = 3.2 +/- 0.8 mu M (SD), 9.0 +/- 3.1 mu M (W)) was attributed to Mrp2-mediated transport, the low affinity component (Km = 192.1 +/- 291.5 mu M (SD), 69.2 +/- 36.2 mu M (W)) may be attributed to transport involving a separate Mrp2 binding site. Data from membranes (Hill coefficient (h) = 2.0 +/- 0.5) and vesicles (h = 1.6 +/- 0.2) expressing Mrp2 and from SD (h = 1.6 +/- 0.4) and Wistar (h = 4.0 +/- 0.6) hepatocytes suggests transport involves more than one binding site. In TR- hepatocytes, CDF efflux was predominantly over the sinusoidal membrane (Km = 100.7 +/- 36.0 mu M), consistent with low abcc2 (Mrp2) expression and compensatory increase in abcc3 (Mrp3) expression. This report shows the potential of using this in vitro method to model changes in biliary excretion due to alterations in transporter expression. (C) 2014 Elsevier Ltd. All rights reserved.

AB - Hepatic efflux of drug candidates is an important issue in pre-clinical drug development. Here we utilise a method which quantifies and distinguishes efflux of drugs at the canalicular and sinusoidal membranes in rat hepatocyte cultures. Bi-phasic kinetics of transport of 5(6)-carboxydichlorofluorescein (CDF) at the canalicular membrane was demonstrated in Sprague Dawley (SD) and Wistar (W) rat hepatocytes. The high affinity component (Km = 3.2 +/- 0.8 mu M (SD), 9.0 +/- 3.1 mu M (W)) was attributed to Mrp2-mediated transport, the low affinity component (Km = 192.1 +/- 291.5 mu M (SD), 69.2 +/- 36.2 mu M (W)) may be attributed to transport involving a separate Mrp2 binding site. Data from membranes (Hill coefficient (h) = 2.0 +/- 0.5) and vesicles (h = 1.6 +/- 0.2) expressing Mrp2 and from SD (h = 1.6 +/- 0.4) and Wistar (h = 4.0 +/- 0.6) hepatocytes suggests transport involves more than one binding site. In TR- hepatocytes, CDF efflux was predominantly over the sinusoidal membrane (Km = 100.7 +/- 36.0 mu M), consistent with low abcc2 (Mrp2) expression and compensatory increase in abcc3 (Mrp3) expression. This report shows the potential of using this in vitro method to model changes in biliary excretion due to alterations in transporter expression. (C) 2014 Elsevier Ltd. All rights reserved.

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KW - membranes and vesicles expressing rat

KW - Mrp2

KW - drug transporters

KW - canalicular efflux

KW - sinusoidal efflux

KW - Dubin-Johnson syndrome

KW - collagen-sandwich configuration

KW - canalicular networks

KW - transport protein

KW - expression

KW - drug

KW - MRP2

KW - membrane

KW - liver

KW - disposition

U2 - 10.1016/j.tiv.2014.05.010

DO - 10.1016/j.tiv.2014.05.010

M3 - Article

VL - 28

SP - 1165

EP - 1175

JO - Toxicology in Vitro

JF - Toxicology in Vitro

SN - 0887-2333

IS - 6

ER -