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.