Role of Rac 1 and cAMP in endothelial barrier stabilization and thrombin-induced barrier breakdown.
Barrier stabilizing effects of cAMP as well as of the small GTPase Rac 1 are well established. Moreover, it is generally believed that permeability-increasing mediators such as thrombin disrupt endothelial barrier functions primarily via activation of Rho A. In this study, we provide evidence that decrease of both cAMP levels and of Rac 1 activity contribute to thrombin-mediated barrier breakdown. Treatment of human dermal microvascular endothelial cells (HDMEC) with Rac 1-inhibitor NSC-23766 decreased transendothelial electrical resistance (TER) and caused intercellular gap formation. These effects were reversed by addition of forskolin/rolipram (F/R) to increase intracellular cAMP but not by the cAMP analogue 8-pCPT-2'-O-Methyl-cAMP (O-Me-cAMP) which primarily stimulates protein kinase A (PKA)-independent signaling via Epac/Rap 1. However, both F/R and O-Me-cAMP did not increase TER above control levels in the presence of NSC-23766 in contrast to experiments without Rac 1 inhibition. Because Rac 1 was required for maintenance of barrier functions as well as for cAMP-mediated barrier stabilization, we tested the role of Rac 1 and cAMP in thrombin-induced barrier breakdown. Thrombin-induced drop of TER and intercellular gap formation were paralleled by a rapid decrease of cAMP as revealed by fluorescence resonance energy transfer (FRET). The efficacy of F/R or O-Me-cAMP to block barrier-destabilizing effects of thrombin was comparable to Y27632-induced inhibition of Rho kinase but was blunted when Rac 1 was inactivated by NSC-23766. Taken together, these data indicate that decrease of cAMP and Rac 1 activity may be an important step in inflammatory barrier disruption.
Baumer
Y
Y
Institute of Anatomy and Cell Biology, University of Würzburg, Würzburg 97070, Germany.
Spindler
V
V
Werthmann
R C
RC
Bünemann
M
M
Waschke
J
J
D000634:Aminoquinolines / Q000494:pharmacology; D015703:Antigens, CD / Q000378:metabolism; D015374:Biosensing Techniques; D015820:Cadherins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002199:Capillary Permeability / Q000187:drug effects; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D017097:Electric Impedance; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D017629:Gap Junctions / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D008856:Microscopy, Fluorescence; D011743:Pyrimidines / Q000494:pharmacology; D020889:Rolipram / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000378:metabolism*; D013997:Time Factors; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000099152:Cadherin 5
D000634:Aminoquinolines; D015703:Antigens, CD; D015820:Cadherins; D002118:Calcium; D005576:Colforsin; D000242:Cyclic AMP; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D011743:Pyrimidines; D020889:Rolipram; D013917:Thrombin; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D000099152:Cadherin 5; C473217:8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate; C487513:NSC 23766; C107953:RAC1 protein, human