P-glycoprotein (P-gp) is an ATP-dependent plasma membrane protein that blocks the cellular influx and/or increases the cellular efflux of a wide variety of unrelated amphipathic compounds. P-gp is constituitively expressed in many tissues including the endothelial cells of the blood-brain-barrier and is encoded for by the MDR1a gene. Although P-gp expression is known to confer a multiple drug resistance phenotype to tumors that express it, an endogenous substrate for P-gp has not been defined. We hypothesized that bilirubin is a substrate for P-gp and that bilirubin transport across the blood-brain-barrier into the central nervous system would be increased in the absence of P-gp expression in brain endothelial capillary cells. We tested this hypothesis using adult MDR1a null mutant transgenic mice (MDR1a[-/-], Cell 77:491-502, 1994). Fifteen MDR1a[-/-] P-gp null mutants and 15 wild type [+/+] mice of the same mixed genetic background were lightly anesthetized and 50 mg/kg of bilirubin infused through the tail vein. Ten or 60 min following infusion the animals were sacrificed, brain vasculature flushed with saline in situ, and brain bilirubin content and serum bilirubin levels determined. Brain bilirubin content (mean±SEM) was significantly higher in MDR1a[-/-] P-gp deficient mice (10.6±1.4 ug/G [n=9]) as compared with wild type [+/+] mice (6.1±0.7 ug/G [n=8]) 10 min following infusion (P<0.05). Brain bilirubin content declined in both groups at the 60 min time point but was higher in MDR1a[-/-] mice (6.2±1.1 ug/G [n=6]) as compared with the wild type [+/+] (3.0±0.5 ug/G [n=7]) (P<0.05). Brain bilirubin clearance, however, did not differ between groups (t1/2 ≈ 55 min). Serum bilirubin levels declined from 10 to 60 min but did not differ between MDR1a [-/-] and [+/+] animals. We conclude that MDR1a[-/-] P-gp deficient mice have significantly higher brain bilirubin content compared with P-gp sufficient controls following an intravenous load of bilirubin. These data suggest that i) bilirubin is a substrate for P-gp and ii) the increased brain bilirubin content in MDR1a[-/-] mice is due to enhanced brain bilirubin influx rather than impaired efflux. We speculate that brain capillary endothelial cell P-gp expression provides a protective effect against bilirubin neurotoxicity by reducing brain bilirubin influx.

Supported by the Magee-Womens Health Foundation Research Fund