Reversible oxidant-induced increases in albumin transfer across cultured endothelium: alterations in cell shape and calcium homeostasis

To determine whether reactive oxygen molecules could directly and reversibly increase the transfer of albumin across an endothelial barrier, we measured albumin transfer across monolayers of endothelium cultured on micropore filters 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 hours in tissue culture media enriched with fetal bovine serum. However, incubation in tissue culture media without serum resulted in progressive injury and cell death. Hence, 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 calcium ionophores, we asked whether oxidants altered endothelial calcium homeostasis. Xanthine-xanthine oxidase increased release of 45Ca++ from preloaded cells. The calcium 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 calcium ionophore A23187 increased endothelial albumin transfer and mimicked the oxidant-induced changes in cell shape. Lanthanum chloride inhibited these effects of A23187. These data suggest that oxygen 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 calcium homeostasis.     

Regulation of hepatic glycogen metabolism: effects of diabetes, insulin infusion, and pancreatic islet transplantation

Insulin-deficient diabetes mellitus results in diminished capacity of the liver to accumulate glycogen. One site of metabolic lesion in the diabetic liver is at the level of the synthase-activating enzyme, synthase phosphatase. This activity is progressively diminished with increasing severity of chemically induced diabetes in both soluble and smooth endoplasmic reticulum (SER) associated subfractions. Insulin administration via an implanted miniosmotic pump or via intrahepatic islet transplantation increased synthase phosphatase activity, particularly in SER. Hepatic glycogen synthesis and accumulation was enhanced as well. The data support a role for insulin in maintenance of the ability of the liver to synthesize and accumulate glycogen mediated either directly or indirectly through SER-synthase phosphatase activity.