THROMBIN-INDUCED RELEASE OF LYSOPHOSPHATIDYLCHOLINE FROM ENDOTHELIAL-CELLS

Lysophosphatidylcholine (LPC) increases extracellularly during ischemi a in vivo in both animals and man as judged by measurements from venou s effluents, but more recent studies have shown little or no increase in buffer-perfused, isolated heart preparations. The appearance of LPC in blood and lymph in animals and in venous effluents in man in respo nse to ischemia suggests a vascular site for the production of LPC. Th e present study was performed to assess whether thrombin could stimula te phospholipase A2 in endothelial cells and whether this would evoke an increase in and release of LPC. Endothelial cells were disassociate d from canine aortas by incubating with 0.1% collagenase for 20 min. C ells were plated and allowed to grow to confluence. Measurement of LPC was performed using Bligh and Dyer extraction of lipids, high perform ance liquid chromatography separation, and quantification of LPC using a recently developed radiometric assay employing [H-3]acetic anhydrid e. Incubation of endothelial cells with thrombin (0.05 unit/ml) result ed in a 2.5-fold increase in LPC to 2.3 +/- 0.1 nmol/mg of protein at 2 min (p < 0.01) and returned to control levels within 20 min. The inc rease in LPC induced by thrombin exhibited a concentration-dependent r esponse with an ED50 = 0.04 unit/ml. A concentration-dependent increas e in LPC was also elicited by stimulation with the peptide portion of the thrombin receptor's tethered ligand SFLLRNPNDKYEPF with an ED50 = 8 muM. The LPC produced was rapidly and completely released into the s urrounding media. Hirudin completely blocked the thrombin-induced incr ease in LPC. Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (0.1 muM) , which rapidly inactivates thrombin's proteolytic activity in situ wi thout impairing binding, or phenyl-prolyl-arginyl-chloromethyl ketone (PPACK, 5 nM), which inactivates thrombin due to chemical alteration o f the proteolytic site, each prevented the increase in LPC in response to thrombin. Stimulation of protein kinase C with phorbol 12-myristat e-13-acetate (PMA, 1 muM) enhanced the response to thrombin. In contra st, staurosporine (100 nM), H7 (15 muM), or chronic treatment with PMA for 20 h to down-regulate protein kinase C completely prevented the i ncrease in LPC in response to thrombin. Thus, thrombin stimulation of endothelial cells in vivo during ischemia may be a primary mechanism c ontributing to the marked increase in LPC extracellularly during ische mia.