Abstract 1348 Poster Session IV, Tuesday, 5/4 (poster 243)

The mdr1a isoform of P-glycoprotein (Pgp) is an ATP dependent integral plasma membrane efflux pump. Pgp is expressed in brain capillary endothelial cells where it limits the influx and CNS retention of a wide variety of unrelated lipophilic compounds. Studies suggest that unconjugated bilirubin is a substrate for Pgp and we have demonstrated that brain bilirubin uptake is significantly increased in adult mdr1a null mutant Pgp deficient transgenic mice as compared with their wild type FVB Pgp sufficient counterparts (Pediatr Res 44:763, 1998). Little is known regarding the ontogeny of Pgp expression in brain microvessels. We examined the expression of Pgp in brain microvessels as a function of maturity from late embryogenesis through adulthood [5 different age groups: embryonic day 16 (E16); day of life 0 (D0); day of life 7 (D7); day of life 21 (D21); and adults] in wild type FVB mice using RT-PCR (n=3 each age) and Western immunoblotting (n=10 each age) techniques. For RT-PCR, 2 ug of mouse brain RNA and bovine retinal RNA (used as an internal control) were converted to cDNA and amplified by PCR using primers directed against mdr1a and bovine rhodopsin. The products were then separated and radioactivity quantitated by phosphorimaging. For Western immunoblots, 20 ug of microvessel protein was used at each developmental age group. The protein was transferred electrophretically onto nitrocellulose and probed with the anti-Pgp monoclonal antibody C219. The relative expression of Pgp was assessed by scanning densitometry. Pgp expression (mean+/-SEM) was indexed as a percent of D0 levels. Pgp mRNA levels increased significantly (p<0.01) as a function of maturation: E16 75+/-8; D21 303+/-37; adult 1160+/-120. Similarly, Pgp protein expression increased significantly (p<0.01) during development: E16 52+/-8; D7 187+/-23; D21 440+/-48; adult 441+/-56. We conclude that Pgp expression in mouse brain microvessels is limited during late embryogenesis and the newborn period and increases markedly with postnatal maturation. In light of evidence that Pgp may play a role in limiting the influx of bilirubin into the CNS, we speculate that the relative low Pgp expression in immature brain may lead to enhanced brain bilirubin uptake.

Supported by Irene McClenahan Young Investigator Research Award (CT), and NIH RO3 HD-35847 (JFW).