BRADYKININ ANTAGONIZES THE EFFECTS OF ALPHA-THROMBIN

alpha-Thrombin (AT) and bradykinin (BK) are endogenous mediators that are released during an inflammatory response, and could have a synergi stic effect on endothelial permeability. Human umbilical vein endothel ial cells (HUVEC) were grown on Transwell membranes and then tested fo r alterations in permeability to fluorescein isothiocyanate-labeled hu man serum albumin. Addition of 1 mu M AT produced a significant increa se in the permeability coefficient at 30 minutes from control levels o f 1.59 x 10(-6) cm/sec to 4.92 x 10(-6) cm/sec. BK (1 mu M) produced a similar increase to 4.46 x 10(-6) cm/sec. For both compounds, permeab ility remained elevated for 90 minutes. Pre-treatment of the HUVEC wit h the bradykinin receptor antagonist, Na-adamantaneacetyl-bradykinin ( NA-BK) (1 mu M), prior to addition of AT, reduced the AT permeability coefficient to 2.69 x 10(-6) cm/sec. Addition of NA-BK (1 mu M) for 5 minutes, then BK (1 mu M) for 5 minutes, inhibited the effect of BK an d of AT (1 mu M) on permeability, decreasing the permeability coeffici ent of the endothelial monolayer to control levels (1.62 x 10(-6) cm/s ec). AT (1 mu M) increased HUVEC intracellular calcium mobilization, a s monitored by FURA-2, to 245 nM from control (70 nM), however, pre-tr eatment with either BK or the bradykinin receptor antagonist decreased the AT induced intracellular calcium mobilization compared to AT alon e. Pre-treatment of the HUVEC with bradykinin (1 mu M) for 2 minutes a lso inhibited the effects of alpha-thrombin (1 mu M) on f-actin distri bution examined by BODIPY-phallodin staining and increased the clottin g times for an alpha-thrombin dependent fibrinogen to fibrin clotting assay. However, incubation of bradykinin (1 mu M) with alpha-thrombin (1 mu M) for either 10 minutes or 100 minutes produced no detectable h ydrolysis products. These data strongly suggest that the inflammatory mediators alpha-thrombin and bradykinin when released together, rather than being synergistic, are antagonistic.