title: Hypoxia/aglycemia increases endothelial permeability: role of second messengers and cytoskeleton.
abstract: The effects of hypoxia/aglycemia on microvascular endothelial permeability were evaluated, and the second messenger systems and the cytoskeletal-junctional protein alterations in this response were also examined. Monolayers of human dermal microvascular endothelial cells on microcarrier beads were exposed to either thioglycolic acid (5 mM, an O(2) chelator), glucose-free medium, or both stresses together. Permeability measurements were performed over a 90-min time course. Although neither hypoxia alone nor aglycemia alone increased endothelial permeability significantly, the combination of both increased significantly as early as 15 min. Intracellular Ca(2+) measurements with fura 2-AM showed that hypoxia/aglycemia treatment increased Ca(2+) influx. To determine the second messengers involved in increased permeability, monolayers were incubated for 30 min with the cytosolic Ca(2+) scavenger 3,4, 5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8, 0.1 mM), a classical protein kinase C (PKC) blocker, Gö-6976 (10 nM), a cGMP-dependent protein kinase (PKG) antagonist, KT-5823 (0.5 microM), or the mitogen-activated protein (MAP) kinase inhibitor PD-98059 (20 microM). Hypoxia/aglycemia-mediated permeability changes were blocked by chelating cell Ca(2+), PKC blockade, PKG blockade, and by inhibiting p38 MAP kinase-1. Finally, changes in the binding of junctional proteins to the cytoskeleton under the same conditions were assessed. The concentrations of occludin and pan-reactive cadherin binding to the cytoskeleton were significantly decreased by only both stresses together. However, these effects were also blocked by pretreatment with TMB-8, Gö-6976, KT-5823 (not in occludin), and PD-98059. These data suggest that hypoxia/aglycemia-mediated endothelial permeability may occur through PKC, PKG, MAP kinase, and Ca(2+) related to dissociation of cadherin-actin and occludin-actin junctional bonds.
authors.affiliation: Department of Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport, Louisiana 71130-3932, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Park
authors.forename: N
authors.initials: N
authors.lastname: Okayama
authors.forename: D
authors.initials: D
authors.lastname: Gute
authors.forename: A
authors.initials: A
authors.lastname: Krsmanovic
authors.forename: H
authors.initials: H
authors.lastname: Battarbee
authors.forename: J S
authors.initials: JS
authors.lastname: Alexander
mesh_terms: D000470:Alkaloids / Q000494:pharmacology; D015820:Cadherins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling / Q000502:physiology; D002227:Carbazoles / Q000494:pharmacology; D015687:Cell Hypoxia / Q000502:physiology; D002463:Cell Membrane Permeability / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000502:physiology*; D020405:Dermis / Q000166:cytology* / Q000201:enzymology; D004727:Endothelium / Q000166:cytology / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007365:Intercellular Junctions / Q000737:chemistry / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008863:Microspheres; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D062793:Occludin; D010100:Oxygen / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D015290:Second Messenger Systems / Q000502:physiology*; D013864:Thioglycolates / Q000494:pharmacology; D015534:Trans-Activators; D051176:beta Catenin
chemical_list: D000470:Alkaloids; C495270:CTNNB1 protein, human; D015820:Cadherins; D002121:Calcium Channel Blockers; D002227:Carbazoles; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007211:Indoles; D007527:Isoenzymes; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D013864:Thioglycolates; D015534:Trans-Activators; D051176:beta Catenin; C073601:KT 5823; C081021:Go 6976; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; D017869:Cyclic GMP-Dependent Protein Kinases; C496628:PRKCA protein, human; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D020928:Mitogen-Activated Protein Kinases; D005947:Glucose; D010100:Oxygen; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium