Adenosine diphosphate-ribosylation of G-actin by botulinum C2 toxin increases endothelial permeability in vitro.

Abstract

The endothelial cytoskeleton is believed to play an important role in the regulation ofendothelial permeability. We used botulinum C2 toxin to perturb cellular actin and determined its effect on the permeability of endothelial cell monolayers derived from porcine pulmonary arteries. The substrate for botulinum C2 toxin is nonmuscle monomeric actin which becomes ADPribosylated. This modified actin cannot participate in actin polymerization and, in addition, acts as a capping protein. Exposure of endothelial cell monolayers to botulinum C2 toxin resulted in a dose-(3-100 ng/ml) and time-dependent (30-120 min) increase in the hydraulic conductivity and decrease in the selecfivity ofthe cell monolayers. The effects ofC2 toxin were accompanied by a time-and dose-dependent increase in ADP-ribosylatin of G-actin. G-Actin content increased and F-actin content decreased time-and dose-dependently in C2 toxin-treated endothelial cells. Phalloidin which stabilizes filamentous actin prevented the effects of botulinum C2 toxin on endothelial permeability. Botulinum C2 toxin induced interendothelial gaps. The effects occurred in the absence of overt cell damage and were not reversible within 2 h. The data suggest that the endothelial microfilament system is important for the regulation ofendothelial permeability. (J. Clin. Invest. 1991. 87:1575-1584

Disruption of cytoskeletal structures mediates shear stress-induced endothelin-1 gene expression in cultured porcine aortic endothelial cells.

“…It is also noteworthy that the dose responses ofthe effects ofphalloidin and cytochalasins on ET-1 gene expression were quite similar to those on G-actin contents in endothelial cells. In addition, the doses of phalloidin and cytochalasins used in this study are not more than those used in the previous literatures ( 15,24). The parallelism between G-actin contents and ET-I mRNA levels and the relevant doses of these agents argue against the possibility that the effects of phalloidin and cytochalasins were not specific for cytoskeletal changes.…”

Autoregulation of Actin Synthesis by Physiological Alterations of the G-Actin Level in Hepatocytes

“…F-actin content was measured by binding of rhodamine-labelled phalloidin to actin filaments (19) in permeabilized and formaldehyde fixed hepatocytes with some modifications (20). Cells were maintained on 6 cm Falcon® culture dishes, washed twice with stabilization buffer (75 mmol/1 KG, 3 mmol/1 MgS 4 , l mmol/1 EGTA, 0.2 mmol/1 dithiothreitol, 0.1 mmol/1 phenylmethylsulphonyl fluoride, 10 mmol/1 imidazole, 10 mg/1 aprotinin, pH 7.2) and permeabilized with 0.3 g/1 saponin in stabilization buffer for 10 min at room temperature.…”

Differential role of actin in lung endothelial and epithelial barrier properties in perfused rabbit lungs

“…The effect of botulinum C 2 toxin on endothelial cell monolayers and human neutrophils is characterized by a time-and dose-dependent increase of G-actin and a decrease of F-actin [24,25]. The kinetics of F-actin loss was compatible with the increase in hydraulic conductivity in the endothelial cell monolayer study [24].…”

“…The effect of botulinum C 2 toxin on endothelial cell monolayers and human neutrophils is characterized by a time-and dose-dependent increase of G-actin and a decrease of F-actin [24,25]. The kinetics of F-actin loss was compatible with the increase in hydraulic conductivity in the endothelial cell monolayer study [24]. As reported previously, intravascular administration of this agent in the isolated lung model provoked a dose-dependent, dramatic increase in the capillary filtration coefficient, concomitant with severe lung oedema formation, both under baseline conditions and, in particular, in response to hydrostatic challenges [12].…”