PASTEURELLA-MULTOCIDA TOXIN INCREASES ENDOTHELIAL PERMEABILITY VIA RHO-KINASE AND MYOSIN LIGHT-CHAIN PHOSPHATASE

Pasteurella multocida toxin (PMT) has been shown to induce actin reorg anization through activation of the GTPase Rho, Here we investigated t he involvement of the Rho target proteins Rho kinase and myosin light chain (MLC) phosphatase in the PMT-induced increase in endothelial per meability and the underlying actin reorganization of endothelial cells . Stimulation of endothelial layers with PMT enhanced transendothelial permeability > 10-fold, and this was abolished by pretreatment with t he specific Rho inactivator C3 transferase from Clostridium botulinum. The PMT-induced increase in endothelial permeability was associated w ith 1) inactivation of MLC phosphatase, 2) an increase in MLC phosphor ylation, and 3) endothelial cell retraction and actin stress fiber for mation, PMT-stimulated actin reorganization could be prevented by 1) p retreatment of cells with C3 transferase, 2) microinjection of the Rho binding domain and the pleckstrin homology domain of Rho kinase, and 3) microinjection of constitutively active MLC phosphatase, Together, these results suggest that PMT activates Rho/Rho kinase, which inactiv ates MLC phosphatase. The resulting increase in MLC phosphorylation ca uses endothelial cell retraction and a rise in endothelial permeabilit y.