cAMP induced Rac 1-mediated cytoskeletal reorganization in microvascular endothelium.
It is well established that cAMP stabilizes endothelial barrier functions, in part by regulation of VE-cadherin via EPAC/Rap 1. The aim of the present study was to investigate whether cAMP activates Rac 1 in microvascular endothelium. In human dermal microvascular endothelial cells (HDMEC), treatment with forskolin/rolipram (F/R) to increase cAMP by as well as the Epac/Rap 1-stimulating cAMP analogue 8-pCPT-2'-O-methyl-cAMP (O-Me-cAMP) stabilized endothelial barrier properties as revealed by raised transendothelial electrical resistance (TER). Under these conditions, immunostaining of VE-cadherin and claudin 5 were increased and linearized. This was paralleled by activation of Rac 1 by 153 +/- 16% (F/R) or 281 +/- 65% (O-Me-cAMP) whereas activity of Rho A was unchanged. F/R and O-Me-cAMP increased the peripheral actin belt and recruited the Rac 1 effector cortactin to cell junctions, similar to direct activation of Rac 1 by CNF-1. Thrombin was used to further test the physiologic relevance of cAMP-mediated Rac 1 activation. Thrombin-induced drop of TER was paralleled by intercellular gap formation, inactivation of Rac 1 and activation of Rho A at 5 and 15 min whereas baseline conditions where re-established following 60 min. Both, F/R and O-Me-cAMP completely blocked the thrombin-induced barrier breakdown. F/R completely abolished thrombin-induced Rac 1 inactivation and Rho A activation whereas O-Me-cAMP only partially blocked Rac 1 inactivation. Taken together, these results indicate that Rac 1 activation likely contributes to the barrier-stabilizing effects of cAMP in microvascular endothelium and that these effects may in part be mediated by Epac/Rap 1.
Baumer
Y
Y
Institute of Anatomy and Cell biology, University of Wuerzburg, Koellikerstrabe 6, 97070, Würzburg, Germany.
Drenckhahn
D
D
Waschke
J
J
D015703:Antigens, CD / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism; D016764:Cell Polarity / Q000187:drug effects; D002478:Cells, Cultured; D062507:Claudin-5; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology*; D003599:Cytoskeleton / Q000502:physiology*; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008833:Microcirculation / Q000166:cytology / Q000502:physiology; D021381:Protein Transport; D020889:Rolipram / Q000494:pharmacology; D012867:Skin / Q000098:blood supply*; D013917:Thrombin / Q000494:pharmacology; D020830:rac1 GTP-Binding Protein / Q000502:physiology*; D020742:rhoA GTP-Binding Protein / Q000502:physiology; D000099152:Cadherin 5
D015703:Antigens, CD; D015820:Cadherins; D062507:Claudin-5; D005576:Colforsin; D000242:Cyclic AMP; D008565:Membrane Proteins; D020889:Rolipram; D013917:Thrombin; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; D000099152:Cadherin 5; C473217:8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate; C107466:CLDN5 protein, human