ROLE OF ENDOTHELIAL CYTOSKELETON IN HIGH-PERMEABILITY EDEMA DUE TO BOTULINUM C-2 TOXIN IN PERFUSED RABBIT LUNGS

The cytoskeleton of the endothelial cell has been suggested to regulate endothelial barrier function. We investigated the role of actin in the maintenance of pulmonary capillary integrity in perfused rabbit lungs. As a tool for selective perturbation of actin, we employed Clostridium botulinum C-2 toxin, which is composed of a membrane translocation component (C-2II) and a component (C-2I) effecting ADP-ribosylation of nonmuscle G-actin. ADP-ribosylated actin is no longer capable of polymerization but acts as a barbed end-capping protein, thereby effecting selective loss of the nonmuscle F-actin content. In buffer-perfused rabbit lungs, combined application of both toxin components (range 50 pg/ml-5 ng/ml C-2I) resulted in a time- and dose-dependent increase in the capillary filtration coefficient (K-fc) With concomitant edema formation. Only 300:600 pg/ml C-2I:II sufficed to induce a > 10-fold rise of K-fc, values within 110 min. This severe lung permeability increase occurred in the absence of vasomotor responses and potassium release or lactate dehydrogenase release. Application of each single toxin component displayed markedly reduced efficacy. Similar to the C-2 toxin effect, severe permeability increase without concomitant hemodynamic changes was evoked by cytochalasin D, known to possess F-actin-disrupting properties. Preloading of lung cells with phallacidin, which in opposition to C-2 toxin decreases F-actin depolymerization, significantly reduced the C-2 toxin-induced increase in vascular permeability. Electron microscopic examination of C-2 toxin-poisoned lungs showed early, extensive endothelial cell attenuations, followed by disruptions of the endothelial layer and marked interstitial edema formation. In contrast, no significant ultrastructural changes of the epithelial type I cells were seen. Collectively, these findings suggest that the actin-based microfilament system plays a crucial role in the maintenance of capillary endothelial cell membrane configuration and barrier function.