Permeability and mechanism of albumin, cationized albumin, and glycosylated albumin transcellular transport across monolayers of cultured bovine brain capillary endothelial cells.
We have measured the permeability and binding characteristics of bovine serum albumin (BSA), cationized BSA (cBSA), and glycosylated BSA (gBSA) to primary cultures of bovine brain capillary endothelial cells (BBCEC). These endothelial cells serve as an in vitro model to study the binding, uptake, and transcellular transport of small and large molecule flux across the blood-brain barrier. The rate of [3H]BSA flux across the cultured BBCEC monolayers grown onto polycarbonate membranes (5-microns pore size) was linear with increasing BSA concentration and the flux could be inhibited by temperature reduction to 0-4 degrees C. The maximal binding of [3H]BSA was 0.04 fmol/mg total cell protein and could not be inhibited by nonradiolabeled BSA. The binding of cBSA and gBSA was rapid and could be inhibited by nonradiolabeled cBSA or gBSA, respectively. The maximal amount bound was 1.8 fmol/mg total cell protein for cBSA and 17.4 fmol/mg total cell protein for gBSA. The dissociation constants (Kd's) were 27 +/- 13 and 3.7 +/- 1.1 nM for cBSA and gBSA, respectively. The flux rates of cBSA and gBSA across the endothelial cell monolayers were linear with respect to concentration and they were approximately seven times greater than those observed for BSA. Each of the proteins appeared on the antiluminal side of the endothelial cell monolayers primarily (90%) as intact protein as determined by trichloroacetic acid (TCA) precipitations and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The results for BSA are similar to those observed for lucifer yellow, a fluid-phase endocytic marker.(ABSTRACT TRUNCATED AT 250 WORDS)
Smith
K R
KR
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Kansas, Lawrence 66045.
Borchardt
R T
RT
D000418:Albumins / Q000378:metabolism*; D000818:Animals; D001812:Blood-Brain Barrier; D002199:Capillary Permeability; D002412:Cations; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D017127:Glycation End Products, Advanced; D007525:Isoelectric Focusing; D012709:Serum Albumin / Q000378:metabolism*; D013696:Temperature; D000093423:Glycated Serum Albumin
D000418:Albumins; D002412:Cations; D017127:Glycation End Products, Advanced; D012709:Serum Albumin; D000093423:Glycated Serum Albumin