Transendothelial permeability of chlorpyrifos in RBE4 monolayers is modulated by astrocyte-conditioned medium

Abstract

The immortalized rat brain endothelium 4 (RBE4) cell line preserves many features of the in vivo brain endothelium. It has been used as an in vitro model of the blood–brain barrier (BBB). Astrocyte–endothelial cell interactions are crucial for maintenance of BBB characteristics. The present study investigated morphological and permeability properties of the RBE4 cell line. Immunohistochemical studies showed positive staining in RBE4 cells for E-cadherin, a Ca 2+-dependent cell–cell adhesion molecule. Western blot immunoassay showed that RBE4 cells consistently express E-cadherin and that its expression significantly increased ( P<0.001) in the presence of astrocyte-conditioned medium (ACM). The transendothelial permeability of chlorpyrifos, an organophosphorus insecticide, was significantly decreased ( P<0.001) when the RBE4 cells were grown in ACM compared with control medium. Additional studies were carried out to determine whether chlorpyrifos is a substrate for the multidrug resistance protein, P-glycoprotein (P-gp). No significant change in chlorpyrifos transendothelial permeability was noted in the presence of verapamil, a P-gp blocker. Thus, in this system, chlorpyrifos is not a substrate for P-gp. This work demonstrates that with additional refinements the RBE4 monolayers might serve as a useful in vitro model for the study of BBB permeability and modulation by astrocyte-derived soluble factors.