Impaired cAMP and Rac 1 signaling contribute to TNF-alpha-induced endothelial barrier breakdown in microvascular endothelium.
OBJECTIVE
In sepsis, tumor necrosis factor-alpha (TNF-alpha) contributes to endothelial barrier breakdown. The involvement of Rho A/rho kinase signaling has recently been challenged. Here, we tested the role of cAMP and Rac 1 signaling.


METHODS
For this study, we took in vivo measurements of hydraulic conductivity in postcapillary mesenteric venules of adult rats. Measurements of transendothelial electrical resistence (TER), fluorescein isothiocyanate-dextran flux, Western blotting, immunostaining, and enzyme-linked immunosorbent assay-based measurements of cAMP levels and Rho-GTPase activity in human microvascular endothelial cells.


RESULTS
TNF-alpha disrupted endothelial barrier functions in vivo and in vitro. Under these conditions, Rho A activity was significantly increased, whereas Rac 1 activity was decreased and Cdc42 was unaltered. Moreover, cAMP levels were reduced. Rho kinase inhibition, using Y27632, did not prevent TNF-alpha-induced barrier breakdown. In contrast, preincubation with forskolin and rolipram (F/R) to increase cAMP and cytotoxic necrotizing factor 1 to activate Rac 1 and Rho A abolished TNF-alpha-induced barrier breakdown in vivo and in vitro. Moreover, inactivation of Rac 1 was blocked by F/R-mediated increase of cAMP, whereas Rho A activation was only partially inhibited.


CONCLUSIONS
Our data indicate that decrease of cAMP and Rac 1 inactivation, rather than Rho A activation, contribute to TNF-alpha-induced endothelial barrier breakdown in vivo and in vitro.
Schlegel
Nicolas
N
University of Wurzburg, Institute of Anatomy and Cell biology, Wurzburg, Germany.
Waschke
Jens
J
D000577:Amides / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D004727:Endothelium / Q000187:drug effects / Q000378:metabolism; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007365:Intercellular Junctions / Q000187:drug effects / Q000378:metabolism; D008233:Lymphotoxin-alpha; D008297:Male; D008833:Microcirculation / Q000187:drug effects* / Q000502:physiology*; D009479:Neuropeptides / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D014699:Venules / Q000187:drug effects / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020742:rhoA GTP-Binding Protein
D000577:Amides; D008233:Lymphotoxin-alpha; D009479:Neuropeptides; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C484602:Rac1 protein, mouse; D011994:Recombinant Proteins; C108830:Y 27632; D000242:Cyclic AMP; D054460:rho-Associated Kinases; C526380:RhoA protein, mouse; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein