Permeability of human venous endothelial cell monolayers perfused in microcarrier cultures: effects of flow rate, thrombin, and cytochalasin D.
We have applied a multiple isotope dilution technique to examine junctional permeability of human umbilical vein endothelial cells (HUVEC) in vitro. Primary cultures were grown to confluence on porous Cytodex-3 microcarrier beads, packed into 0.3 ml columns (3 x 10(6) cells) and perfused at varying flow rates (0.3-1.2 ml/min) with HEPES-buffered Tyrodes solution containing unlabeled cyanocobalamin, insulin, and albumin. Columns were challenged periodically with mixtures of radioactive tracers of different molecular size. Permeability to 22Na+, [57Co]cyanocobalamin (1.3 kD), [125I]insulin (6 kD) or [125I]albumin (66 kD) was assessed relative to [131I]IgG (160 kD, impermeant reference tracer) by comparing column elution profiles. Although the single passage extraction of [125I]albumin by beads alone approximated 40%, the presence of confluent HUVEC rendered these beads effectively impermeable to albumin. High junctional extractions were measured for cyanocobalamin (0.79 +/- 0.02, n = 28) and insulin (0.51 +/- 0.05, n = 14) in cultures perfused at 0.3-0.4 ml/min, and tracer extraction decreased as perfusion rates increased. Permeability coefficients for cyanocobalamin (9.66 x 10(-5) cm/s) and insulin (4.18 x 10(-5) cm/s) increased significantly during perfusion with thrombin (10 U/ml) or cytochalasin D (1 microgram/ml), whereas permeability to albumin (0.39 x 10(-5) cm/s) remained unchanged. Morphological studies, using the glycocalyx stain ruthenium red, revealed that thrombin or cytochalasin D increased the penetration of the stain into junctions between endothelial cells.
Eaton
B M
BM
Department of Obstetrics and Gynaecology, Charing Cross and Westminister Medical School, London, United Kingdom.
Toothill
V J
VJ
Davies
H A
HA
Pearson
J D
JD
Mann
G E
GE
D000418:Albumins / Q000378:metabolism; D002199:Capillary Permeability / Q000187:drug effects; D002478:Cells, Cultured; D015638:Cytochalasin D / Q000494:pharmacology*; D003911:Dextrans; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D008854:Microscopy, Electron; D008863:Microspheres; D011865:Radioisotope Dilution Technique; D012430:Ruthenium Red; D012964:Sodium / Q000378:metabolism; D013194:Staining and Labeling; D013917:Thrombin / Q000494:pharmacology*; D014471:Umbilical Veins; D014805:Vitamin B 12 / Q000378:metabolism
D000418:Albumins; D003911:Dextrans; D007074:Immunoglobulin G; D007328:Insulin; D012430:Ruthenium Red; D015638:Cytochalasin D; C052839:Cytodex; D012964:Sodium; D013917:Thrombin; D014805:Vitamin B 12