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Pharmaceutical Research

, 35:83 | Cite as

Ionophore and Biometal Modulation of P-glycoprotein Expression and Function in Human Brain Microvascular Endothelial Cells

  • Mitchell P. McInerney
  • Irene Volitakis
  • Ashley I. Bush
  • William A. Banks
  • Jennifer L. Short
  • Joseph A. Nicolazzo
Research Paper
  • 171 Downloads

Abstract

Purpose

Biometals such as zinc and copper have been shown to affect tight junction expression and subsequently blood-brain barrier (BBB) integrity. Whether these biometals also influence the expression and function of BBB transporters such as P-glycoprotein (P-gp) however is currently unknown.

Methods

Using the immortalised human cerebral microvascular endothelial (hCMEC/D3) cell line, an in-cell western assay (alongside western blotting) assessed relative P-gp expression after treatment with the metal ionophore clioquinol and biometals zinc and copper. The fluorescent P-gp substrate rhodamine-123 was employed to observe functional modulation, and inductively coupled plasma mass spectrometry (ICP-MS) provided information on biometal trafficking.

Results

A 24-h treatment with clioquinol, zinc and copper (0.5, 0.5 and 0.1 μM) induced a significant upregulation of P-gp (1.7-fold) assessed by in-cell western and this was confirmed with western blotting (1.8-fold increase). This same treatment resulted in a 23% decrease in rhodamine-123 accumulation over a 1 h incubation. ICP-MS demonstrated that while t8his combination treatment had no effect on intracellular zinc concentrations, the treatment significantly enhanced bioavailable copper (4.6-fold).

Conclusions

Enhanced delivery of copper to human brain microvascular endothelial cells is associated with enhanced expression and function of the important efflux pump P-gp, which may provide therapeutic opportunities for P-gp modulation.

KEY WORDS

blood-brain barrier clioquinol ionophore P-glycoprotein transporter 

ABBREVIATIONS

Amyloid beta peptide

AD

Alzheimer’s disease

APS

Ammonium persulfate

BBB

Blood-brain barrier

BMEC

Brain microvascular endothelial cells

CNS

Central nervous system

CQ

Clioquinol

DAPI

4′,6-diamidino-2-phenylindole

EBM2

Endothelial basal medium 2

EDTA

Ethylenediaminetetraacetic acid

HBSS

Hank’s balanced salt solution

hCMEC/D3

Immortalised human cerebral microvascular endothelial cell line

ICP-MS

Inductively coupled plasma mass spectrometry

ICW

In-cell western

IR

Infra-red

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

P-gp

P-glycoprotein

PBS

Phosphate buffered saline

PSC833

Valspodar

R123

Rhodamine-123

SDS

Sodium dodecyl sulfate

TEMED

Tetramethylethylenediamine

TRIS

Tris(hydroxymethyl)aminomethane

WB

Western blot

Notes

Acknowledgments and Disclosures

The studies completed within this publication were funded by the following sources; NHMRC Project APP1048855; the Mason Foundation and the Bethlehem Griffiths Research Foundation. Mitchell P. McInerney is supported by an Australian Government Research Training Program Scholarship.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Mitchell P. McInerney
    • 1
  • Irene Volitakis
    • 2
  • Ashley I. Bush
    • 2
  • William A. Banks
    • 3
    • 4
  • Jennifer L. Short
    • 5
  • Joseph A. Nicolazzo
    • 1
  1. 1.Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences,Monash University,ParkvilleAustralia
  2. 2.The Florey Institute of Neuroscience and Mental Health,ParkvilleAustralia
  3. 3.Division of Gerontology and Geriatric Medicine, Department of Medicine,University of Washington School of MedicineSeattleUSA
  4. 4.Geriatrics Research Education and Clinical Center,Veterans Affairs Puget Sound Health Care System,SeattleUSA
  5. 5.Drug Discovery Biology, Monash Institute of Pharmaceutical SciencesMonash University,ParkvilleAustralia

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