Ionophore and Biometal Modulation of P-glycoprotein Expression and Function in Human Brain Microvascular Endothelial Cells
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.
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.
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).
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 WORDSblood-brain barrier clioquinol ionophore P-glycoprotein transporter
Amyloid beta peptide
Brain microvascular endothelial cells
Central nervous system
Endothelial basal medium 2
Hank’s balanced salt solution
Immortalised human cerebral microvascular endothelial cell line
Inductively coupled plasma mass spectrometry
Phosphate buffered saline
Sodium dodecyl sulfate
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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