VASP is involved in cAMP-mediated Rac 1 activation in microvascular endothelial cells.
Accumulating evidence points to a significant role of vasodilator-stimulated phosphoprotein (VASP) in the maintenance of endothelial barrier functions. We have recently shown that impaired barrier functions in VASP-deficient microvascular myocardial endothelial cells (MyEnd VASP(-/-)) correlated with decreased Rac 1 activity. To further test the hypothesis that VASP is involved in regulation of Rac 1 activity, we studied cAMP-dependent Rac 1 activation. Both inhibition of Rac 1 activation by NSC-23766 and inhibition of PKA by PKI completely blunted the efficacy of forskolin/rolipram (F/R)-mediated cAMP increase to stabilize barrier functions as revealed by measurements of transendothelial resistance (TER). Because these results indicate that PKA/Rac 1 activation is important for barrier stabilization, we tested this signaling pathway in VASP(-/-) cells. We found that F/R and isoproterenol reduced permeability measured as FITC-dextran flux across VASP(-/-) monolayers, but not below baseline levels of wild-type cells (WT). Moreover, cAMP-mediated Rac 1 activation was reduced to approximately 50% of WT levels, and both PKA inhibition by PKI and PKA anchoring via A kinase anchoring peptides (AKAPs) by HT31 almost completely abolished Rac 1 activation in VASP(-/-) and WT endothelium. Accordingly, HT31 significantly reduced F/R-mediated TER increase in WT cells and completely blocked the protective effect of cAMP on endothelial barrier properties. Together, our data underline the significant role of cAMP-mediated Rac 1 activation for endothelial barrier stabilization and demonstrate that both AKAP-mediated PKA anchoring and VASP are required for this process.
Schlegel
Nicolas
N
Institute of Anatomy and Cell Biology, Julius-Maximilians-Univ., Koellikerstr. 6, 97070 Würzburg, Germany.
Waschke
Jens
J
D054758:A Kinase Anchor Proteins / Q000378:metabolism; D000818:Animals; D002199:Capillary Permeability; D015815:Cell Adhesion Molecules / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003911:Dextrans / Q000378:metabolism; D017097:Electric Impedance; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D055806:Microvessels / Q000201:enzymology; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D015290:Second Messenger Systems; D013997:Time Factors; D014162:Transfection; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein; D000098662:Vasodilator-Stimulated Phosphoprotein
D054758:A Kinase Anchor Proteins; D015815:Cell Adhesion Molecules; D003911:Dextrans; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008840:Microfilament Proteins; D009479:Neuropeptides; D010750:Phosphoproteins; C484602:Rac1 protein, mouse; C015219:fluorescein isothiocyanate dextran; D000098662:Vasodilator-Stimulated Phosphoprotein; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D016650:Fluorescein-5-isothiocyanate