Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protection to endothelial barrier dysfunction

Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protection t o endothelial barrier dysfunction. Am. J. Physiol. 277 (Cell Physiol. 46): C580-C588, 1999.-We investigated the hypothesis that cAMP-dependent protein kinase (PKA) protects against endothelial barrier dysfunction in response to proinflammatory mediators. An E1(-), E3(-), replication-deficient adenov irus (Ad) vector was constructed containing the complete sequence of PKA in hibitor (PKI) gene (AdPKI). Infection of human microvascular endothelial ce lls (HMEC) with AdPKI resulted in overexpression of PKI. Treatment with 0.5 phl thrombin increased transendothelial albumin clearance rate (0.012 +/- 0.003 and 0.035 +/- 0.005 mu l/min for control and thrombin, respectively); the increase was prevented with forskolin + 3-isobutyl-1-methylxanthine (F + I) treatment. Overexpression of PKT resulted in abrogation of the F + I- induced inhibition of the permeability increase. However, with HMEC infecte d with ultraviolet-inactivated AdPKI, the F + I-induced inhibition was pres ent. Also, F + I treatment of HMEC transfected with reporter plasmid contai ning the cAMP response element-directed transcription of the luciferase gen e resulted in an almost threefold increase in luciferase activity. Overexpr ession of PKT inhibited this induction of luciferase activity. The results show that Ad-mediated overexpression of PKI in endothelial cells abrogated the cAMP-mediated protection against increased endothelial permeability, pr oviding direct evidence that cAMP-dependent protein kinase promotes endothe lial barrier function.