Hypoxia/aglycemia increases endothelial permeability: role of second messengers and cytoskeleton

Park, J. H., N. Okayama, D. Gute, A. Krsmanovic, H. Battarbee, and J. S. Alexander. Hypoxia/aglycemia increases endothelial permeability: role of second messengers and cytoskeleton. Am. J. Physiol. 277 (Cell Physiol. 46): C1066-C1074, 1999.--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.sub.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.sup.2+] measurements with fura 2-AM showed that hypoxia/aglycemia treatment increased [Ca.sup.2+] influx. To determine the second messengers involved in increased permeability, monolayers were incubated for 30 min with the cytosolic [Ca.sup.2+] scavenger 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8, 0.1 mM), a classical protein kinase C (PKC) blocker, Go-6976 (10 nM), a cGMP-dependent protein kinase (PKG) antagonist, KT-5823 (0.5 [micro]M), or the mitogen-activated protein (MAP) kinase inhibitor PD-98059 (20 [micro]M). Hypoxia/aglycemiamediated permeability changes were blocked by chelating cell [Ca.sup.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, Go-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.sup.2+] related to dissociation of cadherin-actin and occludin-actin junctional bonds. thioglycollate; oxygen; glucose; microvasculature; junction