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

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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.

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Abbreviations

Aβ:

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

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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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Authors and Affiliations

  1. Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences,, Monash University,, 381 Royal Parade, Parkville, VIC,, 3052, Australia

    Mitchell P. McInerney & Joseph A. Nicolazzo

  2. The Florey Institute of Neuroscience and Mental Health,, Parkville, VIC, Australia

    Irene Volitakis & Ashley I. Bush

  3. Division of Gerontology and Geriatric Medicine, Department of Medicine,, University of Washington School of Medicine, Seattle, Washington,, USA

    William A. Banks

  4. Geriatrics Research Education and Clinical Center,, Veterans Affairs Puget Sound Health Care System,, Seattle, Washington,, USA

    William A. Banks

  5. Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University,, Parkville, VIC,, Australia

    Jennifer L. Short

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  1. Mitchell P. McInerney
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Correspondence to Joseph A. Nicolazzo.

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McInerney, M.P., Volitakis, I., Bush, A.I. et al. Ionophore and Biometal Modulation of P-glycoprotein Expression and Function in Human Brain Microvascular Endothelial Cells. Pharm Res 35, 83 (2018). https://doi.org/10.1007/s11095-018-2377-6

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  • DOI: https://doi.org/10.1007/s11095-018-2377-6

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