Endothelin-1 reduces p-glycoprotein transport activity in an in vitro model of human adult blood-brain barrier

Abstract

Aims: It is a huge challenge to understand the blood-brain barrier (BBB), which is a key element in neuroinflammation associated with many brain diseases. The BBB also regulates the passage of xenobiotics into the central nervous system (CNS), and therefore influences drug efficacy. This may be due to the presence of ATP binding cassette transporters such as P-glycoprotein (Pgp) on the BBB, which are efflux pumps known to transport many drugs. The peptide endothelin 1 (ET-1) is involved in different kinds of CNS diseases and neuroinflammation, and is known to modulate Pgp transport activity. Although there are data from animal models, data from human models are scarce. We evaluated Pgp expression and transport activity in adult human brain microvascular endothelial cells (HBMECs) when exposing an adult human in vitro BBB model to ET-1.

Methods: Adult HBMECs were cocultured with human adult glial cells on a Transwells to mimic blood and CNS compartments. These human in vitro BBBs were exposed for 24 h to 100 nM and 10 nM ET-1. Pgp expression was assessed by flow cytometry and its transport activity by measuring radiolabelled digoxin passage.

Results: After exposure to ET-1, flow cytometry showed no shift of fluorescence intensity for a Pgp specific antibody. The passage of digoxin increased with a significant decrease of Q ratio for 10 nM ET-1.

Conclusion: Our results show that ET-1 has no effect on Pgp expression of adult HBMECs, but does modulate Pgp transport activity.

Fig. 1

Pgp expression in adult HBMECs of an adult human in vitro BBB model exposed to ET-1. An adult human autologous in vitro BBB model was exposed to either 10 or 100 nM ET-1 for 24 h in the apical (luminal) compartment. Then, HBMECs were scraped, permeabilised or not and fixed in order to be stained with a specific phycoerythrin-conjugated Pgp antibody (or isotypic control). Pgp expression was then assessed by flow cytometry (in each case, between 5,000 and 10,000 cells were counted). (a) Total Pgp expression in adult permabilised HBMECs of an adult human in vitro BBB model exposed to 100 nM ET-1 for 24 h. (b) Total Pgp expression in adult permabilised HBMECs of an adult human in vitro BBB model exposed to 10 nM ET-1 for 24 h. (c) Pgp expression in adult unpermabilised HBMECs of an adult human in vitro BBB model exposed to either 100 or 10 nM ET-1 for 24 h

Fig. 2

Digoxin passage through a human in vitro BBB model exposed to ET-1. In vitro human adult autologous BBBs were exposed to either 10 nM or 100 nM ET-1 in compartment A for 24 h. [¹⁴C]-Radiolabelled digoxin passage from A (upper chamber) to B (lower chamber) and from B to A compartment was determined in order to calculate the clearance of this substrate. (a) Digoxin clearance from compartment A to B (CL_AB, luminal to abluminal) in a human adult in vitro BBB model exposed to ET-1 for 24 h. (b) Digoxin clearance from compartment B to A (CL_BA, abluminal to luminal) in a human adult in vitro BBB model exposed to ET-1 for 24 h. (c) Digoxin Q ratio compared with control in adult human in vitro BBB after 24-h exposure to ET-1. The ratio Q equals CL_BA/CL_AB