title: Synergistic effects of tumor necrosis factor-alpha and thrombin in increasing endothelial permeability.
abstract: Because activation of the coagulation cascade and the generation of thrombin coexist with sepsis and the release of tumor necrosis factor (TNF)-alpha, we determined the effects of TNF-alpha on the mechanism of thrombin-induced increase in endothelial permeability. We assessed Ca(2+) signaling in human umbilical vein endothelial cells. In human umbilical vein endothelial cells exposed to TNF-alpha for 2 h, thrombin produced a rise in the intracellular Ca(2+) concentration ([Ca(2+)](i)) lasting up to 10 min. In contrast, thrombin alone produced a rise in [Ca(2+)](i) lasting for 3 min, whereas TNF-alpha alone had no effect on [Ca(2+)](i.) Thrombin-induced inositol 1,4,5-trisphosphate generation was not different between control and TNF-alpha-exposed cells. In the absence of extracellular Ca(2+), thrombin produced similar increases in [Ca(2+)](i) in both control and TNF-alpha-exposed cells. In TNF-alpha-exposed cells, the thrombin-induced Ca(2+) influx after intracellular Ca(2+) store depletion was significantly greater and prolonged compared with control cells. Increased Ca(2+) entry was associated with an approximately fourfold increase in Src activity and was sensitive to the Src kinase inhibitor PP1. After TNF-alpha exposure, thrombin caused increased tyrosine phosphorylation of junctional proteins and actin stress fiber formation as well as augmented endothelial permeability. These results suggest that TNF-alpha stimulation of endothelial cells results in amplification of the thrombin-induced Ca(2+) influx by an Src-dependent mechanism, thereby promoting loss of endothelial barrier function.
authors.affiliation: Department of Pharmacology, College of Medicine, University of Illinois at Chicago, 60612, USA. tiruc@uic.edu
authors.forename: C
authors.initials: C
authors.lastname: Tiruppathi
authors.forename: T
authors.initials: T
authors.lastname: Naqvi
authors.forename: R
authors.initials: R
authors.lastname: Sandoval
authors.forename: D
authors.initials: D
authors.lastname: Mehta
authors.forename: A B
authors.initials: AB
authors.lastname: Malik
mesh_terms: D000418:Albumins / Q000493:pharmacokinetics; D002118:Calcium / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004357:Drug Synergism; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006490:Hemostatics / Q000494:pharmacology*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014471:Umbilical Veins / Q000166:cytology; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D000418:Albumins; D006490:Hemostatics; D014409:Tumor Necrosis Factor-alpha; D015544:Inositol 1,4,5-Trisphosphate; D019061:src-Family Kinases; D013917:Thrombin; D002118:Calcium