Relationship of oxidant-mediated cytotoxicity to phospholipid metabolism in endothelial cells.
Exposure to oxidants permeabilizes cell membranes and liberates unesterified fatty acids (UFA) in a variety of cell types, including endothelial cells. Products of phospholipase activity, particularly UFA and lysophosphatides, possess potent detergent-like properties, and we postulated that oxidant injury might be mediated by the accumulation of these toxic phospholipase products. Several radiolabels were incorporated into defined positions in the phospholipids of cultured, confluent bovine pulmonary endothelial cells (BPAEC). The release of radiolabeled fatty acids and the accumulation of cell-associated phospholipase products were measured and compared to a standard cytotoxicity assay (51Cr release) in response to an oxidant stress, in this case 0.1 to 10 mM hydrogen peroxide (H2O2). H2O2 caused time- and dose-dependent 51Cr release as well as liberation of saturated ([14C]stearic acid) and unsaturated ([3H]arachidonic acid) fatty acids and the accumulation of phospholipase A2 and C products. The ability of BPAEC to incorporate UFA into complex phospholipids was shown to be severely impaired in the presence of H2O2. Further studies showed that H2O2 caused depletion of BPAEC adenosine triphosphate (ATP) content to undetectable levels, and that the depletion of cellular ATP by iodoacetic acid induced substantial release of [3H]arachidonic acid but not [14C]stearic acid from BPAEC. This finding suggests that release of UFA in response to an oxidant stress may be due in part to a defect in ATP-dependent reacylation pathways and need not reflect any increase in phospholipase activities. Also unsaturated fatty acids were found to be toxic to BPAEC upon adding them to supernatants of cultured monolayers.
Duane
P G
PG
Pulmonary Section, Veterans Administration Medical Center, Minneapolis, MN 55417.
Rice
K L
KL
Charboneau
D E
DE
King
M B
MB
Gilboe
D P
DP
Niewoehner
D E
DE
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002860:Chromium Radioisotopes; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D015227:Lipid Peroxidation; D010740:Phospholipases / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011651:Pulmonary Artery / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D013229:Stearic Acids / Q000378:metabolism
D001095:Arachidonic Acids; D002860:Chromium Radioisotopes; D010743:Phospholipids; D013229:Stearic Acids; C031183:stearic acid; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D010740:Phospholipases