MECHANISMS OF CHOLERA-TOXIN PREVENTION OF THROMBIN-INDUCED AND PMA-INDUCED ENDOTHELIAL-CELL BARRIER DYSFUNCTION

Thrombin-induced endothelial cell (EC) activation leads to compromise of monolayer barrier function due to cellular retraction/contraction a nd intercellular gap formation. Cyclic AMP induces relaxation in other contractile cells and promotes barrier function in EC. To investigate mechanisms involved in cAMP protection in thrombin-induced permeabili ty, we pretreated bovine pulmonary arterial EC monolayers with 1 mu g/ ml cholera holotoxin which catalyzed ADP ribosylation of G(s) and incr eased synthesis of cAMP. The holotoxin, but not the binding subunit, r educed basal permeability and prevented gap formation and permeability following challenge with 1 mu M thrombin, 100 mu M thrombin receptor- activating peptide, or 1 mu M phorbol myristate acetate (PMA). Further more, thrombin-induced gap formation and permeability were reversed by cholera toxin post-treatment. Pretreatment with 5 mu M forskolin or 1 mM dibutyryl cAMP, with or without 1 mM isobutyl methylxanthine, but not cGMP analogs, protected against thrombin-induced EC permeability, mimicking the cholera toxin effect. Although downregulation of protein kinase C attenuated both thrombin- and PMA-induced permeability, chol era toxin did not alter either PMA-induced protein kinase C activation or thrombin-induced Ca2+ mobilization. In contrast, cholera toxin att enuated thrombin-induced myosin light chain phosphorylation and largel y prevented actin redistribution. These studies suggest that cholera t oxin: (1) protects endothelial barrier function and reverses establish ed dysfunction via increased cAMP, (2) does not alter thrombin recepto r interaction or early signal events such as Ca2+ mobilization and PKC activation, (3) attenuates myosin light chain kinase activation and a ctomyosin contractile interaction subsequent to thrombin activation, a nd (4) abrogates contractile processes subsequent to PKC activation, w hich is also an important mechanism in thrombin-induced permeability b ut is independent of myosin light chain kinase activation. (C) 1994 Ac ademic Press, Inc.