PKA inhibits RhoA activation: a protection mechanism against endothelial barrier dysfunction.
Much evidence indicates that cAMP-dependent protein kinase (PKA) prevents increased endothelial permeability induced by inflammatory mediators. We investigated the hypothesis that PKA inhibits Rho GTPases, which are regulator proteins believed to mediate endothelial barrier dysfunction. Stimulation of human microvascular endothelial cells (HMEC) with thrombin (10 nM) increased activated RhoA (RhoA-GTP) within 1 min, which remained elevated approximately fourfold over control for 15 min. The activation was accompanied by RhoA translocation to the cell membrane. However, thrombin did not activate Cdc42 or Rac1 within similar time points, indicating selectivity of activation responses by Rho GTPases. Pretreatment of HMEC with 10 micro M forskolin plus 1 micro M IBMX (FI) to elevate intracellular cAMP levels inhibited both thrombin-induced RhoA activation and translocation responses. FI additionally inhibited thrombin-mediated dissociation of RhoA from guanine nucleotide dissociation inhibitor (GDI) and enhanced in vivo incorporation of (32)P by GDI. HMEC pretreated in parallel with FI showed >50% reduction in time for the thrombin-mediated resistance drop to return to near baseline and inhibition of approximately 23% of the extent of resistance drop. Infection of HMEC with replication-deficient adenovirus containing the protein kinase A inhibitor gene (PKA inhibitor) blocked both the FI-mediated protective effects on RhoA activation and resistance changes. In conclusion, the results provide evidence that PKA inhibited RhoA activation in endothelial cells, supporting a signaling mechanism of protection against vascular endothelial barrier dysfunction.
Qiao
Jing
J
Department of Pharmacology, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612, USA.
Huang
Fei
F
Lum
Hazel
H
D000256:Adenoviridae / Q000235:genetics; D002199:Capillary Permeability / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D015398:Signal Transduction / Q000502:physiology; D012867:Skin / Q000098:blood supply; D013917:Thrombin / Q000494:pharmacology; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D002352:Carrier Proteins; D006490:Hemostatics; D047908:Intracellular Signaling Peptides and Proteins; C015482:protein kinase modulator; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D013917:Thrombin; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein