Thrombin stimulates the release of arachidonate but not 8,11,14-eicosatrienoate from endothelial cell glycerolipids

Previous studies in their laboratory have shown that thrombin stimulates the release of arachidonate (20:4(n-6)) but not 22:4(n-6) from endothelial cell glycerolipids. The authors now report that thrombin also does not significantly stimulate the release of 8,11,14-[14C]eicosatrienoate per se. Human umbilical vein endothelial cells were radiolabeled for 24 hr with 1.25 μM [14C]20:3(n-6) or [14C]20:4(n-6). When incubated for 10 min in buffered saline with 50 μM fat-free albumin and 1 U/ml thrombin, these cells released 4.1% and 7.6%, respectively, of the previously incorporated radioactivity. Analysis of released 14C-fatty acids by radio-gas chromatography indicated that the thrombin-stimulated release from cells prelabelled with [14C]20:3(n-6) was essentially due to release of [14C]arachidonate synthesized endogenously by desaturation of the [14C]20:3(n-6). Expressed as a percentage of each 14C-fatty acyl present moiety in cellular glycerolipids of cells prelabelled with [14C]20:3(n-6), release was 8.2% for arachidonate but only 0.63% for 20:3(n-6). Studies with other 14C-fatty acids indicate that 5,8,11-20:3 is released in response to thrombin (5-9%); 8,11,14,17-20:4 is not (<1%). These results suggest that a Δ5 double bond in the fatty acid is necessary for thrombin-stimulated release from endothelial cell glycerolipids