THROMBIN AND BRADYKININ INITIATE DISCRETE ENDOTHELIAL SOLUTE PERMEABILITY MECHANISMS

This study documents the discrete solute permeability mechanisms associated with physiologically high concentrations of human alpha-thrombin and bradykinin stimulation of bovine pulmonary artery endothelial cell (BPAEC) monolayers using fluorescein isothiocyanate-hydroxyethyl starch macromolecules. Agonist-induced alterations of intracellular free calcium ([Ca2+]i) using fura-2 acetoxymethyl ester were also measured. BPAEC monolayers showed restricted diffusion consistent with a small-pore (approximately 150 angstrom) radius under baseline conditions. Thrombin produced a major increase in monolayer permeability that was greatest for solute molecular radii (a(e)) >100 angstrom. This effect was associated with the exposure of the large (approximately 2,000 angstrom) pores of the filter support by 50- to 1,050-mum2 open areas between approximately 0.5% of the adjacent endothelial cells. This heterogeneous endothelial barrier of parallel large- and small-pore transport pathways permitted solute convection with free diffusion across a few large pores to dominate the restricted diffusion of most apparently unperturbed endothelial junctions. Bradykinin produced a small, transient elevation in monolayer permeability to a(e) <35 angstrom, consistent with an increase in the number of small pores or a decrease in path length of this transport pathway. The bradykinin- and thrombin-induced peak elevations in [Ca2+]i were inversely associated with the degree of increased monolayer solute permeability, and enzymatically inhibited thrombin produced none of these effects. These data show that bradykinin and human alpha-thrombin represent two distinct classes of endothelial cell agonists that initiate discrete solute permeability mechanisms.