Glucosylation of small GTP-binding Rho proteins disrupts endothelial barrier function.
The endothelial cytoskeleton is important for the regulation of endothelial barrier function. Small GTP-binding Rho proteins play a central role in the organization of the microfilament system. Clostridium difficile toxin B (TcdB) inactivates Rho proteins by glucosylation at Thr-37. We used TcdB as a probe to study the role of Rho proteins in the regulation of endothelial barrier function. TcdB time (50-170 min) and dose (10-100 ng/ml) dependently increased the hydraulic conductivity of cultured porcine pulmonary artery endothelial cell monolayers approximately 10-fold. Simultaneously, the albumin reflection coefficient decreased substantially from 0.8 to 0.15. Before endothelial hyperpermeability, TcdB reduced F-actin content in a dose-dependent manner, whereas G-actin content remained unchanged. Finally, we proved that TcdB caused dose (5-100 ng/ml)- and time-dependent glucosylation of Rho proteins in endothelial cells. Phalloidin, which stabilizes filamentous actin, prevented the effect of TcdB on endothelial permeability. In contrast to thrombin-, hydrogen peroxide-, or Escherichia coli hemolysin-induced hyperpermeability, the elevation of cyclic nucleotides did not block TcdB-related permeability. The data demonstrate a central role of small GTP-binding Rho proteins for the control of endothelial barrier function.
Hippenstiel
S
S
Department of Internal Medicine, Justus-Liebig-University, Giessen, Germany.
Tannert-Otto
S
S
Vollrath
N
N
Krüll
M
M
Just
I
I
Aktories
K
K
von Eichel-Streiber
C
C
Suttorp
N
N
D000199:Actins / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D009712:Nucleotides, Cyclic / Q000502:physiology; D013552:Swine; D014532:Uridine Diphosphate Glucose / Q000494:pharmacology
D000199:Actins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D009712:Nucleotides, Cyclic; C031815:toxB protein, Clostridium difficile; D019204:GTP-Binding Proteins; D005947:Glucose; D014532:Uridine Diphosphate Glucose