J. Mchowat et Pb. Corr, THROMBIN-INDUCED RELEASE OF LYSOPHOSPHATIDYLCHOLINE FROM ENDOTHELIAL-CELLS, The Journal of biological chemistry, 268(21), 1993, pp. 15605-15610
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.