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 (125)I-albumin in RLMVEC monolayers occurred at a rate of 7.2 fmol. min(-1). 10(6) cells(-1). Albumin transport was inhibited by cell surface depletion of the 60-kDa albumin-binding glycoprotein gp60 and by disruption of caveolae using methyl-beta-cyclodextrin. By contrast, gp60 activation (by means of gp60 cross-linking using primary and secondary antibodies) increased (125)I-albumin uptake 2.3-fold. At 37 degrees C, (125)I-albumin uptake had a half time of 10 min and was competitively inhibited by unlabeled albumin (IC(50) = 1 microM). Using a two-site model, we estimated by Scatchard analysis the affinity (K(D)) and maximal capacity (B(max)) of albumin uptake to be 0.87 microM (K(D1)) and 0.47 pmol/10(6) cells (B(max1)) and 93.3 microM (K(D2)) and 20.2 pmol/10(6) cells (B(max2)). At 4 degrees C, we also observed two populations of specific binding sites, with high (K(D1) = 13.5 nM, 1% of the total) and low (K(D2) = 1.6 microM) affinity. On the basis of these data, we propose a model in which the two binding affinities represent the clustered and unclustered gp60 forms. The model predicts that fluid phase albumin in caveolae accounts for the bulk of albumin internalized and transported in the endothelial monolayer.
John
Theresa A
TA
Department of Pharmacology, College of Medicine, University of Illinois, 835 S. Wolcott Avenue, Chicago, IL 60612, USA.
Vogel
Stephen M
SM
Tiruppathi
Chinnaswamy
C
Malik
Asrar B
AB
Minshall
Richard D
RD
D000818:Animals; D001667:Binding, Competitive; D001692:Biological Transport; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D011652:Pulmonary Circulation; D051381:Rats; D012709:Serum Albumin / Q000493:pharmacokinetics*; D012795:Sialoglycoproteins / Q000502:physiology
D012709:Serum Albumin; D012795:Sialoglycoproteins; C065610:sialoglycoprotein gp60