Time course of thrombin-induced increase in endothelial permeability: Relationship to Ca(i)/²⁺ and inositol polyphosphates

The temporal relationship between the α-thrombin-induced increase in transendothelial permeability and the α-thrombin-mediated changes in several key transmembrane signaling events was examined in confluent monolayers of bovine pulmonary artery endothelial cells (BPAEC). The time courses of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] generation, changes in cytosolic [Ca²⁺] ([Ca²⁺](i)), and reorganization of cytoskeletal F-actin were determined to assess the relationship between these events and the onset of the α-thrombin-induced increase in endothelial permeability. α-Thrombin (10^-7 M) increased the transendothelial ¹²⁵I-albumin clearance rate half-maximally by ~1 min and maximally by ~2 min (160% over control level). The increase in permeability occurred concomitantly with reorganization of F- actin cytoskeleton (i.e., loss of peripheral band and increased stress fiber density) and increased actin polymerization. Stimulation of fura-2-loaded BPAEC with 10^-7 M α-thrombin produced a typical biphasic rise in [Ca²⁺](i). The initial rapid increase in [Ca²⁺](i) peaked by ~16 s after thrombin challenge and the [Ca²⁺](i) response showed a slow decrease to half-maximal within 50 s. The α-thrombin-induced increase in permeability as well as the increase in [Ca²⁺](i) were consistently preceded by increased Ins(1,4,5)P₃ generation detectable within 10 s after thrombin challenge. These results indicate that α-thrombin triggers a cascade of events {i.e., Ins(1,4,5)P₃ generation and the ensuing rise in [Ca²⁺](i)}, which may comprise the second messengers that mediate F-actin reorganization and the increase in endothelial permeability.