Journal
AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY
Volume 284, Issue 6, Pages L972-L980Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajplung.00429.2002
Keywords
Rho guanosine 5 '-triphosphate; protein kinase A inhibitor; guanine nucleotide dissociation inhibitor; endothelial resistance
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Funding
- NHLBI NIH HHS [HL-62649] Funding Source: Medline
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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 muM forskolin plus 1 muM 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 P-32 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 similar to23% 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.
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