Antioxidant enzyme content of pulmonary artery endothelial cells: effects of subculture.
The activities of glutathione peroxidase, superoxide dismutase and catalase, enzymes which play a critical role in protection of the vascular endothelium from oxygen free-radical injury, were determined in large vessel endothelial cells obtained under three different growth conditions: from freshly isolated from bovine pulmonary arteries, in the first (primary) subculture and after six serial subcultures (6.5 population doublings). The endothelium was obtained by mechanically scraping the vascular lumen. Endothelial cell monolayers were detached mechanically from the substratum prior to passage. No proteolytic enzymes were used in either procedure. The activities of catalase, superoxide dismutase and glutathione peroxidase determined in freshly isolated endothelial cells were, respectively, 39.9 +/- 10.3, 2.2 +/- 0.8 and 3.0 +/- 0.5 X 10(2) units per mg protein. After primary culture there was no change in superoxide dismutase activity, but a significant decrease in glutathione peroxidase activity to 1.4 +/- 0.4 X 10(2) was observed, and catalase activity dropped significantly to 18.6 +/- 5.0 units per mg protein. After 6.5 population doublings, the activity of all three enzymes returned to values similar to those of the freshly isolated cells. A fourfold increase in the protein to DNA ratio occurred in cells in primary culture and was maintained in sixth-passage cells. This increase in endothelial cell size upon culture was reflected in the electron microscopic evidence of cellular hypertrophy. Measurement of the rate of transport of 5-hydroxytryptamine by endothelial cell monolayers revealed a substantial loss upon multiple passage. Transport in the sixth-passage cells was decreased to one-half the rate of primary cells.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002374:Catalase / Q000032:analysis*; D002417:Cattle; D002478:Cells, Cultured; D004727:Endothelium / Q000166:cytology / Q000201:enzymology / Q000648:ultrastructure; D005979:Glutathione Peroxidase / Q000032:analysis*; D008854:Microscopy, Electron; D011651:Pulmonary Artery / Q000166:cytology / Q000201:enzymology* / Q000648:ultrastructure; D012701:Serotonin / Q000378:metabolism; D013482:Superoxide Dismutase / Q000032:analysis*
D012701:Serotonin; D002374:Catalase; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase