Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protection to endothelial barrier dysfunction.
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 adenovirus (Ad) vector was constructed containing the complete sequence of PKA inhibitor (PKI) gene (AdPKI). Infection of human microvascular endothelial cells (HMEC) with AdPKI resulted in overexpression of PKI. Treatment with 0.5 microM thrombin increased transendothelial albumin clearance rate (0.012 +/- 0.003 and 0.035 +/- 0.005 microl/min for control and thrombin, respectively); the increase was prevented with forskolin + 3-isobutyl-1-methylxanthine (F + I) treatment. Overexpression of PKI resulted in abrogation of the F + I-induced inhibition of the permeability increase. However, with HMEC infected with ultraviolet-inactivated AdPKI, the F + I-induced inhibition was present. Also, F + I treatment of HMEC transfected with reporter plasmid containing the cAMP response element-directed transcription of the luciferase gene resulted in an almost threefold increase in luciferase activity. Overexpression of PKI 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, providing direct evidence that cAMP-dependent protein kinase promotes endothelial barrier function.
Lum
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Department of Pharmacology, Rush-Presbyterian-St. Luke's Medical Center, University of Illinois, Chicago, Illinois 60612, USA. hlum@rush.edu
Jaffe
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Schulz
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Masood
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RayChaudhury
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Green
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D000256:Adenoviridae / Q000235:genetics; D000257:Adenoviridae Infections / Q000378:metabolism; D002199:Capillary Permeability / Q000502:physiology*; D002352:Carrier Proteins / Q000235:genetics*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology; D004730:Endothelium, Vascular / Q000378:metabolism*; D015870:Gene Expression / Q000502:physiology*; D017930:Genes, Reporter / Q000502:physiology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D014158:Transcription, Genetic / Q000502:physiology; D019076:Transgenes / Q000502:physiology
D002352:Carrier Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D047908:Intracellular Signaling Peptides and Proteins; C015482:protein kinase modulator; D000242:Cyclic AMP