Transcytosis of albumin in endothelial cells is brefeldin A--independent.
To determine whether in endothelial cells (EC) the pathways of endocytosis and transcytosis of macromolecules interconnect, the effect of Brefeldin A (BFA) on these processes was tested. To this purpose EC were grown to confluence on plastic culture dishes or on cell culture chamber inserts placed into corresponding wells, so as to obtain a dual chamber system. The cells maintained the typical characteristics of EC and had an electrical resistance in the range of 30-60 Ohm.cm2. Transendothelial transport of albumin conjugated to the fluorochrom Texas Red (Alb-TR) and of horseradish peroxidase (HRP) added to the upper compartment, in the absence or presence of BFA (0-25 micrograms/ml), was evaluated in aliquots collected from the lower compartment. At different time intervals, quantitative data were obtained by fluorimetry and spectrophotometry. In other experiments transcytosis of Alb-TR was examined in the presence of 100 microM forskolin (an inhibitor of BFA effect). The endocytosis of Alb-TR and HRP was evaluated by incubating EC with the probes, and the internalized tracers determined in the cell lysate using the methods described above. The results showed that BFA has no significant effect on transcytosis of albumin and HRP. In contradistinction, BFA (5 micrograms/ml) reduced markedly endocytosis of HRP (by 47%). Forskolin has no effect on transcytosis. The data indicate that the BFA-induced perturbance in the endocytic route does not affect the transcytotic pathway of albumin, and suggest that in EC, transcytosis of macromolecules may represent a shortcut for rapid and direct transport of some plasma molecules across the cell.
Antohe
F
F
Institute of Cellular Biology and Pathology Nicolae Simionescu, Bucharest, Romania.
Serban
G
G
Radulescu
L
L
Simionescu
M
M
D000818:Animals; D001011:Aorta; D001692:Biological Transport / Q000187:drug effects; D020126:Brefeldin A; D002417:Cattle; D018929:Cell Culture Techniques / Q000295:instrumentation; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D003517:Cyclopentanes / Q000494:pharmacology*; D004705:Endocytosis / Q000187:drug effects*; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005456:Fluorescent Dyes; D006735:Horseradish Peroxidase; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D014966:Xanthenes
D003517:Cyclopentanes; D005456:Fluorescent Dyes; D014966:Xanthenes; D005576:Colforsin; D020126:Brefeldin A; D012710:Serum Albumin, Bovine; C034657:Texas red; D006735:Horseradish Peroxidase