Quantitative analysis of albumin uptake and transport in the rat microvessel endothelial monolayer

We determined the concentration dependence of albumin binding, uptake, and transport in confluent monolayers of cultured rat lung microvascular endothelial cells (RLMVEC). Transport of 125I-albumin in RLMVEC monolayers occurred at a rate of 7.2 fmol · min−1 · 106cells−1. Albumin transport was inhibited by cell surface depletion of the 60-kDa albumin-binding glycoprotein gp60 and by disruption of caveolae using methyl-β-cyclodextrin. By contrast, gp60 activation (by means of gp60 cross-linking using primary and secondary antibodies) increased 125I-albumin uptake 2.3-fold. At 37°C, 125I-albumin uptake had a half time of 10 min and was competitively inhibited by unlabeled albumin (IC50= 1 μM). Using a two-site model, we estimated by Scatchard analysis the affinity (K D) and maximal capacity (Bmax) of albumin uptake to be 0.87 μM (K D1) and 0.47 pmol/106 cells (Bmax1) and 93.3 μM (K D2) and 20.2 pmol/106 cells (Bmax2). At 4°C, we also observed two populations of specific binding sites, with high (K D1 = 13.5 ...