We have previously shown that the main source of arachidonate in thrombin-stimulated human platelets is 1-acyl-2-arachidonoyl (AA) glycerophosphocholine (GPC) and release of 3H-AA from this phospholipid also was correlated with increased 3H-AA in ether phospholipid. This ATP independent transfer of 3H-AA from 1,2 diacyl GPC to ether phospholipid (transacylation) also occurs in resting cells. Human platelets in 1/10 volume of plasma (ACD anticoagulant, pH 6.5) were radiolabelled with 3H-AA for 60 min at 37°C and then exogenous 3H-AA was removed by gel filtration into Tyrode's buffer, pH 7.4, 0.2% albumin. These radiolabelled cells were incubated in the absence of exogenous 3H-AA for four hours followed by Bligh and Dyer extraction and thin layer chromatography purification of phospholipids. 3H-AA in 1,2 diacyl GPC was found to decrease by over 20% and increase substantially in 1-0-alkyl-2-acyl GPC and 1-0-alk-1'-enyl-2-acyl glycerophospho ethanolamine (GPE), In this same time interval the mass of AA released by thrombin (5 U/ml, 10 min, 37°C, no stirring)in the presence of BIT 775C and measured by GLC, stayed the same (30 nmoles/109 cells), however, the specific activity decreased. Using reverse phase HPLC to resolve diradylglycerobenzoate derivatives of phospholipids: acylation, deacylation, and transacylation were observed for individual AA-containing molecular species of phospholipid, including those with an unsaturated fatty acid at sn-1. In particular the radiolabellinq of the 1-unsaturate-2-arachidonoyl GPC correlated with the specific activity of the 3H-AA released by stimulation with thrombin. Furthermore, 1-arachidonoyl-2-3H-arachidonoyl GPC was completely deacylated while 50 % of its mass remained. This contrasted with 16:0, and 18:0-2-arachidonoyl GPC in which the specific activity remained the same before and after deacylation. We conclude that deacylation of AA-containing molecular species of 1,2 diacyl GPC in stimulated cells includes molecular species which are also a source of arachidonic acid for transacylation reactions.
Shotgun Lipidomics Identifies a Paired Rule for the Presence of Isomeric Ether Phospholipid Molecular Species
