REVERSIBLE OXIDANT-INDUCED INCREASES IN ALBUMIN TRANSFER ACROSS CULTURED ENDOTHELIUM - ALTERATIONS IN CELL-SHAPE AND CALCIUM HOMEOSTASIS

Abstract

To determine whether reactive O2 molecules could directly and reversibly increase the transfer of albumin across an endothelial barrier, albumin transfer across monolayers of [porcine] endothelium cultured on micropore filters was measured before and after exposure to xanthine and xanthine oxidase. Xanthine and xanthine oxidase increased endothelial albumin transfer in a dose-dependent fashion. Parallel phase contrast and fluorescence microscopy demonstrated retraction of adjacent cells from one another and disruption of the actin filaments. The oxidant-induced increases in albumin transfer and changes in cell shape were reversed by removing xanthine oxidase and then incubating the monolayers for 3 1/2 h in tissue culture media enriched with fetal bovine serum. Incubation in tissue culture media without serum resulted in progressive injury and cell death. The brief exposure to oxidants initiated a progressive injury process that was reversed by incubation in serum. Because intracellular and extracellular calcium are important determinants of cell shape, and because some oxidized membrane lipids act as Ca ionophores, whether oxidants altered endothelial calcium homeostasis was questioned. Xanthine-xanthine oxidase increased release of 45Ca2 from preloaded cells. The Ca antagonist lanthanum chloride prevented xanthine-xanthine oxidase increases in endothelial albumin transfer and prevented the changes in cell shape; chelation of extracellular calcium inhibited lysis of endothelium by xanthine-xanthine oxidase; and the Ca ionophore A23187 increased endothelial albumin transfer and mimicked the oxidant-induced changes in cell shape. Lanthanum chloride inhibited these effects of A23187. O2 radicals can reversibly increase endothelial permeability to macromolecules that this is associated with reversible changes in endothelial cell shape and actin filaments, and that the changes in cell shape are related to oxidant-induced changes in endothelial Ca homeostasis.