Mechanisms of cholera toxin prevention of thrombin- and PMA-induced endothelial cell barrier dysfunction.

Abstract

Thrombin-induced endothelial cell (EC) activation leads to compromise of monolayer barrier function due to cellular retraction/contraction and 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 permeability, we pretreated bovine pulmonary arterial EC monolayers with 1 microgram/ml cholera holotoxin which catalyzed ADP ribosylation of Gs and increased synthesis of cAMP. The holotoxin, but not the binding subunit, reduced basal permeability and prevented gap formation and permeability following challenge with 1 microM thrombin, 100 microM thrombin receptor-activating peptide, or 1 microM phorbol myristate acetate (PMA). Furthermore, thrombin-induced gap formation and permeability were reversed by cholera toxin post-treatment. Pretreatment with 5 microM 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, cholera toxin did not alter either PMA-induced protein kinase C activation or thrombin-induced Ca2+ mobilization. In contrast, cholera toxin attenuated thrombin-induced myosin light chain phosphorylation and largely prevented actin redistribution. These studies suggest that cholera toxin: (1) protects endothelial barrier function and reverses established dysfunction via increased cAMP (2) does not alter thrombin receptor interaction or early signal events such as Ca2+ mobilization and PKC activation, (3) attenuates myosin light chain kinase activation and actomyosin contractile interaction subsequent to thrombin activation, and (4) abrogates contractile processes subsequent to PKC activation, which is also an important mechanism in thrombin-induced permeability but is independent of myosin light chain kinase activation.