Bilirubin Efflux By Brain Capillary Endothelial Cell Monolayers in Vitro: Role of P-Glycoprotein.

Abstract

Background: The passage of bilirubin across the blood-brain barrier into the CNS is central to the development of kernicterus. Indirect in vivo evidence suggests that P-glycoprotein (Pgp), a multidrug transporter expressed on brain capillary endothelial cells, may limit the influx and CNS retention of unconjugated bilirubin (Pediatr Res 44:763–766, 1998). This phenomenon has not been studied in brain capillary endothelial cell monolayers in vitro. Objective: To test the hypothesis that Pgp mediates bilirubin transport across brain capillary endothelial cell monolayers in vitro. Design/Methods: Bovine brain capillary endothelial cells (Cell Systems Corp.) were grown in confluent monolayers at a density of 5 × 104 cells/insert in 1 cm2 Transwell dishes. [3H]-bilirubin (100nM) transport (pnol/cm2/min) was tested in both the apical to basolateral [A→B] and basolateral to apical [B→A] directions in the presence and absence of a Pgp inhibitor (cyclosporin A[5uM]). Involvement of a Pgp mediated efflux mechanism is suggested by a B→A/A→B ratio of greater than 1.5 (Pharm Res 16:1206, 1999). Brain capillary endothelial cell Pgp expression was confirmed by Western immunoblotting techniques. Results: Brain capillary endothelial cells express Pgp as seen on Western immunoblots. Bilirubin transport in the B→A direction (0.67±0.05 pmol/cm2/min) was 6.4 fold higher than the rate for A→B direction (0.11±0.02) suggesting active efflux of bilirubin across brain capillary endothelial cell monlolayers. B→A bilirubin transport decreased (0.60±0.07) and A→B bilirubin transport was enhanced (0.17±0.03) in the presence of the Pgp inhibition, with an overall decrease in bilirubin efflux of 27% suggesting that bilirubin transport by brain capillary endothelial cells is mediated in part by Pgp. Conclusions: We conclude that i) bilirubin is transported by brain capillary endothelial cell monolayers in vitro in a net B→A direction (i.e. activie efflux); ii) Pgp plays an active role in this barrier function, and iii) unconjugated bilirubin is a substrate for Pgp. We speculate that i) brain capillary endothelial Pgp limits the CNS passage and retention of unconjugated bilirubin and ii) that low brain capillary endothelial cell Pgp expression, as reported in premature neonates, may enhance brain bilirubin levels during hyperbilirubinemia. Disclosure: Supported by NINDS (038993), the 25 Club of Magee-Womens Hospital and the Mario Lemieux Centers for Patient Care and Research.