Lysophosphatidylcholine (LPC) increases extracellularly during
ischemia in vivo in both animals and man as judged by measurements from venous 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 response to
ischemia suggests a vascular site for the production of LPC. The present study was performed to assess whether
thrombin could stimulate
phospholipase A2 in endothelial cells and whether this would evoke an increase in and release of LPC. Endothelial cells were disassociated from canine aortas by incubating with 0.1%
collagenase for 20 min. Cells were plated and allowed to grow to confluence. Measurement of LPC was performed using Bligh and Dyer extraction of
lipids, high performance liquid chromatography separation, and quantification of LPC using a recently developed radiometric assay employing [3H]
acetic anhydride. Incubation of endothelial cells with
thrombin (0.05 unit/ml) resulted 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 increase in LPC induced by
thrombin exhibited a concentration-dependent response with an ED50 = 0.04 unit/ml. A concentration-dependent increase in LPC was also elicited by stimulation with the
peptide portion of the
thrombin receptor's tethered
ligand SFLLRNPNDKYEPF with an ED50 = 8 microM. The LPC produced was rapidly and completely released into the surrounding media.
Hirudin completely blocked the
thrombin-induced increase in LPC.
Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (0.1 microM), which rapidly inactivates
thrombin's proteolytic activity in situ without impairing binding, or phenyl-prolyl-arginyl-chloromethyl
ketone (
PPACK, 5 nM), which inactivates
thrombin due to chemical alteration of the proteolytic site, each prevented the increase in LPC in response to
thrombin. Stimulation of
protein kinase C with
phorbol 12-myristate-13-acetate (PMA, 1 microM) enhanced the response to
thrombin. In contrast,
staurosporine (100 nM), H7 (15 microM), or chronic treatment with PMA for 20 h to down-regulate
protein kinase C completely prevented the increase in LPC in response to
thrombin. Thus,
thrombin stimulation of endothelial cells in vivo during
ischemia may be a primary mechanism contributing to the marked increase in LPC extracellularly during
ischemia.