MECHANISMS OF ALPHA-THROMBIN, HISTAMINE, AND BRADYKININ-INDUCED ENDOTHELIAL PERMEABILITY

alpha-Thrombin, bradykinin, and histamine are endogenous mediators tha t increase endothelial permeability. We examined the mechanism by whic h these three vasoactive mediators could alter permeability to albumin of human umbilical vein endothelial cells (HUVEC). HUVEC were grown t o confluence on Transwell membranes and we monitored the flux of fluor escein isothiocyanate-labeled human serum albumin across the membrane from the upper to lower chamber of the Transwell. Addition of alpha-th rombin, bradykinin, or histamine increased the permeability coefficien t of the HUVEC monolayer. At 30 min the permeability coefficient for a lpha-thrombin was 4.92 x 10(-6) cm/sec while histamine was 4.47 x 10(- 6) cm/sec. Maximum changes in the permeability coefficient were about threefold control baseline values (1.59 x 10(-6) cm/sec). There was al so a temporal difference in the magnitude of the permeability coeffici ent. alpha-Thrombin and bradykinin induced HUVEC permeability was incr eased for the first 90 min after which it returned to control levels. In contrast, histamine increased the permeability of the HUVEC monolay er throughout the 2 h experiment. To determine a possible intracellula r mechanism of the altered permeability coefficients, HUVEC were label ed with FURA-2 and intracellular calcium was monitored by digital fluo rescence ratio imaging. Maximum intracellular calcium in HUVEC was inc reased by alpha-thrombin (245 +/- 20 nM) and histamine (210 +/- 22 nM) , but not by bradykinin (70 +/- 7 nM) as compared to control (69 +/- 1 0). Fluorescent photomicrographs of HUVEC stimulated with the three ag onists indicated that alpha-thrombin and histamine substantially alter ed HUVEC f-actin arrangement, while bradykinin had no effect on HUVEC f-actin distribution. These data support previous in vitro and in vivo studies demonstrating increased permeability by all three agonists. T hese data also show, for the first time, that histamine and alpha-thro mbin increased permeability by calcium-dependent intracellular pathway s, but bradykinin operates through a calcium-independent mechanism. (C ) 1996 Wiley-Liss, Inc.