Phosphatidylcholine metabolism in endothelial cells: evidence for phospholipase A and a novel Ca2+-independent phospholipase C.
The metabolism of phosphatidylcholine (PC) was investigated in sonicated suspensions of bovine pulmonary artery endothelial cells and in subcellular fractions using two PC substrates: 1-oleoyl-2-[3H]oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phospho[14C]choline. When these substrates were incubated with the whole cell sonicate at pH 7.5, all of the metabolized 3H label was recovered in [3H]oleic acid (95%) and [3H]diacylglycerol (5%). All of the 14C label was identified in [14C]lysoPC (92%) and [14C]phosphocholine (8%). These data indicated that PC was metabolized via phospholipase(s) A and phospholipase C. Substantial diacylglycerol lipase activity was identified in the cell sonicate. Production of similar proportions of diacylglycerol and phosphocholine and the low relative activity of phospholipase C compared to phospholipase A indicated that the phospholipase C-diacylglycerol lipase pathway contributed little to fatty acid release from the sn-2 position of PC. Neither phospholipase A nor phospholipase C required Ca2+. The pH profiles and subcellular fractionation experiments indicated the presence of multiple forms of phospholipase A, but phospholipase C activity displayed a single pH optimum at 7.5 and was located exclusively in the particulate fraction. The two enzyme activities demonstrated differential sensitivities to inhibition by p-bromophenacylbromide, phenylmethanesulfonyl fluoride and quinacrine. Each of these agents inhibited phospholipase A, whereas phospholipase C was inhibited only by p-bromophenacylbromide. The unique characteristics observed for phospholipase C activity towards PC indicated the existence of a novel enzyme that may play an important role in lipid metabolism in endothelial cells.
D000818:Animals; D002417:Cattle; D003902:Detergents / Q000494:pharmacology; D004075:Diglycerides / Q000378:metabolism; D004727:Endothelium / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008071:Lipoprotein Lipase / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D003902:Detergents; D004075:Diglycerides; D010713:Phosphatidylcholines; D010740:Phospholipases; D010741:Phospholipases A; D008071:Lipoprotein Lipase; D010738:Type C Phospholipases; C017251:1,2-oleoylphosphatidylcholine; C013965:diolein