Department of Pathology, St. Louis University School of Medicine,
St. Louis, Missouri 63104
The present
study was performed to characterize thrombin-stimulated phospholipase
A2
(PLA2) activity and the
resultant release of lysophospholipids from endothelial cells. The
majority of PLA2 activity in
endothelial cells was membrane associated,
Ca2+ independent, and arachidonate
selective. Incubation with thrombin increased membrane-associated
PLA2 activity using both
plasmenylcholine and alkylacyl glycerophosphocholine substrates in the
absence of Ca2+, with no increase
in activity observed with phosphatidylcholine substrate. The increased
PLA2 activity was accompanied by
arachidonic acid and lysoplasmenylcholine (LPlasC) release from
endothelial cells into the surrounding medium. Thrombin-induced changes
were duplicated by stimulation with the thrombin-receptor-directed peptide SFLLRNPNDKYEPF. Pretreatment with the
Ca2+-independent
PLA2 inhibitor bromoenol lactone
blocked thrombin-stimulated increases in
PLA2 activity, arachidonic acid,
and LPlasC release. Stimulation of protein kinase C (PKC) increased
basal PLA2 activity and LPlasC
production. Thrombin-stimulated
PLA2 activity and LPlasC production were enhanced with PKC activation and completely prevented with PKC downregulation. Thus thrombin treatment of endothelial cells
activates a PKC-activated, membrane-associated,
Ca2+-independent
PLA2 that selectively hydrolyzes
arachidonylated, ether-linked phospholipid substrates, resulting in
LPlasC and arachidonic acid release.
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INTRODUCTION |
DURING BRIEF INTERVALS OF myocardial ischemia,
choline lysophospholipids [lysophosphatidylcholine (LPC) and
lysoplasmenylcholine (LPlasC)] increase in venular and lymphatic
effluents from the ischemic zone in laboratory animals (1, 26) and in
coronary venous effluents from patients with pacing-induced
ischemia (25). The increase in choline lysophospholipids in the
extracellular space during the first few minutes of ischemia
occurs before any evidence of irreversible cell damage, suggesting that
the appearance in blood and lymph may originate, at least in part, from
a vascular site. The accumulation of choline lysophospholipids plays an
important role in the development of ischemia-induced
ventricular arrhythmias (14, 20). It is interesting to note, however,
that although addition of micromolar concentrations of LPC to the
extracellular buffer exerts dramatic electrophysiological alterations
(14), intracellular microinjection of LPC into isolated cardiac
myocytes has no effect on electrophysiological properties (1).
Accordingly, extramyocytic production of LPC is likely to play an
important role in the induction of electrophysiological abnormalities
during myocardial ischemia.
Thrombotic occlusion of a major coronary artery at a site of
preexisting atherosclerosis is the underlying cause of the vast majority of myocardial infarctions (6). It has been demonstrated that
the incidence of ventricular arrhythmias is greater when ischemia results from intracoronary thrombus formation as
opposed to balloon occlusion (8), suggesting that products released from or associated with an intracoronary thrombus may directly or
indirectly influence the electrophysiological properties of cardiac
myocytes. Although multiple factors in thrombus formation may play a
role in arrhythmogenesis, we demonstrated previously that thrombin
increases the production and release of choline lysophospholipids in
endothelial cells (16) at thrombin concentrations much lower than those
measured adjacent to evolving thrombi (23). These observations suggest
that a direct link may exist between evolving thrombi in the coronary
circulation and the initiation of arrhythmogenesis as a result of
increased production by endothelial cells of choline lysophospholipids
that become incorporated into the ischemic cardiac myocyte sarcolemma
and initiate electrophysiological derangements.
The majority of studies in endothelial cells to date inferred an
increase in phospholipase A2
(PLA2) activity in response to
thrombin stimulation by measuring the increase in one of the metabolites of PLA2 hydrolysis,
such as arachidonic acid or prostacyclin. However, activation of
PLA2 is not the only mechanism
present in cells that can lead to arachidonic acid production, and thus caution must be used in inferring activation of a particular enzymatic pathway when measuring arachidonic acid or eicosanoid production alone.
In this study, we show that thrombin stimulation of endothelial cells
results in activation of a membrane-associated,
Ca2+-independent
PLA2 that is selective for
ether-linked arachidonylated phospholipid substrates. Activation of
this PLA2 isoform results in
increased LPlasC, with no corresponding increase in LPC, and an
increase in arachidonic acid release. An increase in these metabolites
in the ischemic myocardium may lead to the production of arrhythmias
soon after the onset of myocardial ischemia.
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METHODS |
Isolation and culture of endothelial cells.
Aortas were removed from adult pigs and placed in buffer containing (in
mmol/l) 117 NaCl, 1.2 CaCl2, 5.3 KCl, 26 NaHCO3, 1.0 NaH2PO4,
and 5.6 glucose (pH 7.4). All branching arteries were ligated, and the
lumen was washed with fresh buffer, filled with additional buffer
containing 1 mg/ml collagenase (type II; Worthington), and incubated at
37°C for 20 min. The effluent was collected and centrifuged at 350 g for 10 min. The endothelial cell
pellet was resuspended in medium 199 with Earle's salts (Sigma
Chemical) containing 20% fetal bovine serum (Life Technologies), 50 µg/ml endothelial cell growth supplement (Collaborative Research), 30 µg/ml streptomycin, and 30 U/ml penicillin (Sigma Chemical), and aliquots were added to 100-mm culture dishes (coating C, MatTek). Nonadherent cells and debris were removed by changing the medium after 24 h. Cells were then allowed to grow to confluence, achieving a
contact-inhibited monolayer of flattened, closely opposed endothelial cells in 6-7 days.
Confluent monolayers were stained immunocytochemically with a mouse
monoclonal antibody directed against von Willebrand antigen (American
Diagnostica). More than 95% of cells expressed detectable von
Willebrand antigen.
Stimulation of confluent endothelial cells.
In all cases, experiments were carried out with the endothelial cells
present as a confluent monolayer. Before the experiment, confluent
endothelial cells were washed with HEPES buffer composed of
(in mmol/l) 133.5 NaCl, 4.8 KCl, 1.2 MgCl2, 1.2 KH2PO4,
10 HEPES, 10 glucose, and 1.2 CaCl2 (pH 7.4).
Thrombin, SFLLRNPNDKYEPF peptide (SFLL), and
FSLLRNPNDKYEPF peptide (FSLL) were dissolved in HEPES buffer
at a stock concentration 100× the final concentration used. As
appropriate, bromoenol lactone (BEL), methyl arachidonyl fluorophosphonate (MAFP), mepacrine, and dibucaine were dissolved in
HEPES buffer and added before thrombin stimulation. Phorbol 12-myristate 13-acetate (PMA) was prepared in DMSO at a concentration of 1 mM and diluted in HEPES buffer immediately before use.
Measurement of PLA2 activity.
After stimulation, the surrounding HEPES buffer was removed and
replaced with ice-cold buffer containing (in mmol/l) 250 sucrose, 10 KCl, 10 imidazole, 5 EDTA, and 2 dithiothreitol, with 10% glycerol (pH
7.8). Cells were removed from the culture dishes with a rubber policeman, placed on ice, and sonicated for 10 s. When appropriate, subcellular fractions from endothelial cells were isolated by centrifuging the sonicate at 14,000 g
for 10 min to remove unbroken cells, nuclei, and mitochondria and then
centrifuging the supernatant at 100,000 g for 60 min to separate cytosolic and
membrane fractions. PLA2 activity
was assessed by incubating enzyme (20 µg membrane protein, 200 µg
cytosolic protein, or 75 µg total protein) with 100 µM
plasmenylcholine, phosphatidylcholine, or alkylacyl
glycerophosphocholine substrates radiolabeled with oleic acid
(16:0,[3H]18:1) or
arachidonic acid
(16:0,[3H]20:4) at the
sn-2 position (substrate composition
is represented as
a:b,c:d,
where
a:b
and
c:d
represent the chain length:no. of double bonds for the aliphatic groups
at the sn-1 and
sn-2 positions, respectively, of the
corresponding phospholipid substrate molecule). The substrate was
introduced into the incubation mixture by injection in ethanol (5 µl). Incubations were performed in assay buffer containing 100 mmol/l
Tris and 10% glycerol (pH 7.0) with either 4 mmol/l EGTA or 10 mmol/l
Ca2+ at 37°C for 5 min in a
total volume of 200 µl. Reactions were terminated by the addition of
100 µl butanol, vortexed, and centrifuged at 2,000 g for 5 min. Released radiolabeled
fatty acid was isolated by TLC and quantified by liquid scintillation
spectrometry. These reaction conditions resulted in linear reaction
velocities with respect to both time and enzyme concentration for each
substrate examined. The 100 µM substrate concentration was selected
to ensure that maximal reaction velocities were obtained and to ensure
negligible isotope dilution effects by endogenous substrate. To define
PLA2 specific activity, total
PLA2 activity was normalized to
protein content measured as described by Markwell et al. (15) with the use of lyophilized BSA as the protein standard.
Synthesis of radiolabeled plasmenylcholine, phosphatidylcholine,
and alkylacyl glycerophosphocholine for PLA2
activity measurements.
Radiolabeled plasmenylcholine, phosphatidylcholine, or alkylacyl
glycerophosphocholine substrates for assessment of
PLA2 activity were prepared by
reacting unlabeled 16:0 LPlasC, 16:0 LPC, or 16:0
lyso-platelet-activating factor (LPAF), respectively, with radiolabeled
fatty anhydride utilizing
N,N-dimethyl-4-aminopyridine as a catalyst as described previously (17). Radiolabeled fatty anhydride was prepared from
[9,10-3H]oleic acid or
[5,6,8,9,11,12,14,15-3H(N)]arachidonic
acid utilizing dicyclohexylcarbodiimide-mediated condensation of the
fatty acid. Radiolabeled products were purified by passing the reaction
mixture through an amine solid-phase extraction column,
followed by HPLC using Partisil SCX. Unlabeled 16:0 LPlasC was isolated
and purified from bovine heart choline phospholipids as described
previously (5). Unlabeled 16:0 LPC and LPAF were purchased from Sigma Chemical.
Measurement of choline lysophospholipids.
LPC and LPlasC measurements were made using a modification of a
radiometric assay method described previously (7, 17). The procedure
involves the extraction of lipids from the endothelial cells by the
method of Bligh and Dyer (3), followed by the separation of the
lysophospholipids from other phospholipids by HPLC. The purified LPC
and LPlasC fractions, as well as known amounts of LPC and LPlasC
standards, were then acetylated with [3H]acetic anhydride
using 0.33 M dimethylaminopyridine as a catalyst. The acetylated
lysophospholipid was then separated by TLC, and radioactivity was
quantified by liquid scintillation spectrometry. Standard curves were
constructed, and LPC and LPlasC levels were derived for all samples and
normalized to the protein content of the endothelial cells as described
previously (15).
[14C]LPC was added as
an internal standard to all samples to correct for loss of sample that
occurred during extraction, purification, and acetylation. Recoveries
were consistently >80%.
Measurement of arachidonic acid release.
Arachidonic acid release was determined by measuring
[3H]arachidonic acid
released into the surrounding medium from the endothelial cell
monolayer previously labeled with
[3H]arachidonic acid.
Briefly, endothelial cells were incubated at 37°C with 3 µCi
[3H]arachidonic
acid/culture dish for 18 h. This incubation resulted in >70%
incorporation of radioactivity into the endothelial cells. Eighty-five
percent of incorporated radioactivity was recovered from
phosphatidylcholine or phosphatidylethanolamine phospholipids. After
incubation, endothelial cell monolayers were washed three times with
Tyrode solution containing 3.6% BSA to remove unincorporated [3H]arachidonic acid.
Endothelial cells were incubated at 37°C for 15 min before
experimental conditions. At the end of the stimulation period, the
surrounding medium was removed to a scintillation vial and represented
the amount of radiolabeled arachidonic acid released from the
endothelial cells during the stimulation interval. The amount of
radiolabeled arachidonic acid remaining in the endothelial cell
monolayer was measured by adding 1 ml of 10% SDS, removing the cells
from the culture well by scraping, and adding them to a scintillation
vial. These samples were left overnight to allow total solubilization
of the endothelial cell protein before the addition of liquid
scintillant. Radioactivity in both surrounding medium and endothelial
cells was quantified by liquid scintillation spectrometry.
Statistical analysis.
Statistical comparisons were performed using Student's unpaired
t-test or, as appropriate, ANOVA
followed by Fishers least significant difference test to compare
individual means. All results are expressed as means ± SE.
P < 0.05 was considered
statistically significant.
 |
RESULTS |
PLA2 activity was measured in
subcellular fractions isolated from porcine endothelial cells using
plasmenylcholine, phosphatidylcholine, and alkylacyl
glycerophosphocholine radiolabeled with oleic acid or arachidonic acid
at the sn-2 position in the absence or
presence of Ca2+.
PLA2 activity in membrane and
cytosolic subcellular fractions measured under control or
thrombin-stimulated (0.05 U/ml, 1 min) conditions is shown in Table
1. The majority of endothelial cell PLA2 activity in unstimulated
cells is membrane associated, Ca2+
independent, and selective for arachidonylated phospholipid substrates (Table 1). Thrombin stimulation resulted in a significant increase in
membrane-associated PLA2 activity
measured using plasmenylcholine and alkylacyl glycerophosphocholine
substrates (Table 1). No increase in membrane-associated
PLA2 activity measured using
phosphatidylcholine was detected (Table 1). No change in cytosolic
PLA2 activity was measured
following thrombin stimulation. Thus thrombin stimulation of
endothelial cells results in an increase in membrane-associated, calcium-independent PLA2 activity
that is selective for phospholipid substrates with an ether linkage at
the sn-1 position and arachidonate at
the sn-2 position.
After characterization of PLA2
activity in endothelial cells with respect to subcellular localization,
Ca2+ dependency, and substrate
selectivity, we subsequently measured PLA2 activity using whole cell
sonicates and
(16:0,[3H]18:1)
phospholipid substrates in the absence of
Ca2+. Endothelial cells were
incubated with thrombin (0.05 U/ml) for selected intervals up to 20 min
(Fig. 1). Thrombin
stimulation caused a significant increase in
PLA2 activity measured using both
(16:0,[3H]18:1)
plasmenylcholine and
(16:0,[3H]18:1)
alkylacyl glycerophosphocholine (Fig. 1) after 30 s of stimulation. PLA2 activity
measured with plasmenylcholine remained elevated for 2 min, whereas
that measured with alkylacyl glycerophosphocholine returned to basal
levels following 1 min of thrombin stimulation (Fig. 1). In contrast,
no change in PLA2 activity was
observed using
(16:0,[3H]18:1)
phosphatidylcholine over the total incubation time (Fig. 1).

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Fig. 1.
Time course of phospholipase A2
(PLA2) activity measured in
endothelial cells following stimulation with 0.05 U/ml thrombin.
PLA2 activity was measured by
incubating 75 µg endothelial cell protein with 100 µmol/l
(16:0,[3H]18:1)
plasmenylcholine ( ),
(16:0,[3H]18:1)
alkylacyl glycerophosphocholine ( ), or
(16:0,[3H]18:1)
phosphatidylcholine ( ) in absence of
Ca2+ (4 mM EGTA) at 37°C for 5 min. Values are means ± SE of independent results derived from
endothelial cells isolated from 4 animals.
* P < 0.05, ** P < 0.01 compared with
control values.
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Because thrombin stimulation of endothelial cells resulted in
activation of a Ca2+-independent
PLA2 that is selective for
ether-linked phospholipids, it would be expected that accompanying the
increased PLA2 activity there
would be a corresponding increase in lysophospholipids and free fatty
acid produced by the cleavage of plasmalogen phospholipids in the
membrane. We have adapted a previously published method of
lysophospholipid measurement (8, 13) to enable us to measure LPlasC
(the lysophospholipid produced following cleavage of plasmenylcholine) and LPC (the lysophospholipid produced following cleavage of
phosphatidylcholine) separately. After thrombin stimulation, we
observed an increase in LPlasC content in endothelial cells after 1 min, which remained elevated up to 5 min of stimulation and then
returned to basal levels (Fig. 2). No
corresponding increase in LPC was measured (Fig. 2).
Accompanying the increase in LPlasC observed in response to thrombin
stimulation, we observed a threefold increase in release of free
[3H]arachidonic acid
after 2 min of thrombin stimulation (from 1.9 ± 0.1 to 6.1 ± 0.2%, P < 0.01, n = 6), which remained elevated for 20 min before returning to basal levels.

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Fig. 2.
Time course for production of lysoplasmenylcholine (LPlasC) and
lysophosphatidylcholine (LPC) following stimulation of endothelial
cells with thrombin (0.05 U/ml). Values are means ± SE of
independent results from endothelial cells isolated from 4 animals.
** P < 0.01 compared with
control values.
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Endothelial cells were incubated with increasing thrombin
concentrations for 1 min, and the concentration response curve for thrombin-induced changes in PLA2
activity is shown in Fig. 3. PLA2 activity using
(16:0,[3H]18:1)
plasmenylcholine was significantly increased with thrombin concentrations >0.01 U/ml (Fig. 3). The half-maximal
effective dose (ED50) for
plasmenylcholine substrate was 0.06 U/ml thrombin. Significant
increases in PLA2 activity using
alkylacyl glycerophosphocholine were observed at thrombin
concentrations >0.05 U/ml (Fig. 3). No significant change in
PLA2 activity was observed using
phosphatidylcholine at any concentration of thrombin used (Fig. 3). A
significant increase in LPlasC was observed with concentrations of
thrombin >0.01 U/ml, with maximal increases in LPlasC at thrombin
concentrations >0.1 U/ml (Fig. 4). The
ED50 for LPlasC production was
0.03 U/ml. No corresponding increase in LPC was observed at any
thrombin concentration (Fig. 4).

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Fig. 3.
Concentration response curve for
PLA2 activity measured in
endothelial cells in response to stimulation with thrombin for 1 min.
PLA2 activity was measured by
incubating 75 µg endothelial cell protein with 100 µmol/l
(16:0,[3H]18:1)
plasmenylcholine ( ), (16:0,
[3H]18:1) alkylacyl
glycerophosphocholine ( ), or
(16:0,[3H]18:1)
phosphatidylcholine ( ) in absence of
Ca2+ (4 mM EGTA) at 37°C for 5 min. Values are means ± SE of independent results derived from
endothelial cells isolated from 5 animals.
* P < 0.05, ** P < 0.01 compared with
control values.
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Fig. 4.
Concentration response curve for production of LPlasC and LPC following
stimulation of endothelial cells with thrombin for 1 min. Values are
means ± SE of independent results from endothelial cells isolated
from 4 animals. ** P < 0.01 compared with control values.
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The amino terminus of the thrombin receptor is cleaved by thrombin
between residues Arg-41 and Ser-42, exposing a new amino terminus with
the peptide sequence SFLLRNPNDKYEPF (SFLL) that serves as a tethered
ligand (27). The SFLL peptide has been demonstrated to stimulate the
thrombin receptor and elicit responses similar to those seen following
thrombin stimulation in a variety of cells (16). Stimulation of
confluent endothelial cells with increasing concentrations of SFLL for
1 min resulted in a concentration-dependent increase in
PLA2 activity using both
(16:0,[3H]18:1)
plasmenylcholine and (16:0,[3H]18:1)
alkylacyl glycerophosphocholine (Fig. 5) at
SFLL concentrations >0.5 µM. These increases are similar to those
observed with thrombin (Fig. 3). No increase in
PLA2 activity was observed with
(16:0,[3H]18:1)
phosphatidylcholine (Fig. 5). Bypassing thrombin's proteolytic activity and directly stimulating the thrombin receptor with the agonist peptide thus elicits increases in
PLA2 activity similar to those in
response to thrombin stimulation. At the same concentrations, incubation of endothelial cells with the peptide FSLL resulted in no
increase in PLA2 activity (data
not shown). Thus receptor stimulation is highly specific for the
receptor-directed peptide, since transposition of the first two amino
acids on the peptide resulted in complete loss of receptor activation.
Accompanying the increase in PLA2
activity observed with SFLL stimulation of endothelial cells, a
concentration-dependent increase in LPlasC, with no increase in LPC,
was observed with increasing SFLL concentrations (Fig.
6).

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Fig. 5.
Concentration response curve for
PLA2 activity measured in
endothelial cells in response to stimulation with
SFLLRNPNDKYEPF peptide (SFLL) for 1 min.
PLA2 activity was measured by
incubating 75 µg endothelial cell protein with 100 µmol/l
(16:0, [3H]18:1)
plasmenylcholine ( ),
(16:0,[3H]18:1)
alkylacyl glycerophosphocholine ( ), or
(16:0,[3H]18:1)
phosphatidylcholine ( ) in absence of
Ca2+ (4 mM EGTA) at 37°C for 5 min. Values are means ± SE of independent results derived from
endothelial cells isolated from 5 animals.
** P < 0.01 compared with
control values.
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Fig. 6.
Concentration response curve for production of LPlasC and LPC following
stimulation of endothelial cells with SFLL for 1 min. Values are means ± SE of independent results from endothelial cells isolated from 4 animals. ** P < 0.01 compared
with control values.
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To determine whether LPlasC was released into the surrounding medium or
remained cell associated, LPlasC content and LPC content were measured
in the medium and endothelial cell monolayer separately. After thrombin
stimulation (0.05 U/ml, 1 min), the surrounding medium was removed from
the endothelial cell monolayer, and phospholipids were extracted
separately from each sample before lysophospholipid quantitation.
Thrombin stimulation of endothelial cells resulted in an increase in
LPlasC in the medium, with no corresponding increase in LPlasC in the
endothelial cells (Fig. 7). No increase in
LPC was observed in either sample. Thus thrombin stimulation of
endothelial cells results in a release of LPlasC into the surrounding medium of the endothelial cell monolayer.

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Fig. 7.
Release of LPlasC into surrounding medium following stimulation with
thrombin (0.05 U/ml, 1 min). LPlasC associated with endothelial cells
was not significantly increased by thrombin stimulation (open bars).
Increase in LPlasC following thrombin stimulation is a measurement of
release of LPlasC into surrounding medium (hatched bars). No
corresponding increase in LPC was observed. Values are means + SE of
independent results from endothelial cells isolated from 6 animals.
** P < 0.01 compared with
corresponding control values.
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BEL is a selective inhibitor of
Ca2+-independent
PLA2 (11). Incubation of
endothelial cells with BEL (10 µM, 10 min) reduced basal
PLA2 activity measured using
(16:0,[3H]18:1)
plasmenylcholine and
(16:0,[3H]18:1)
alkylacyl glycerophosphocholine (Fig.
8). No significant decrease in basal
PLA2 activity was measured with
(16:0,[3H]18:1)
phosphatidylcholine substrate by BEL pretreatment (Fig. 8).
Pretreatment of endothelial cells with BEL before thrombin stimulation
(0.05 U/ml, 1 min) resulted in complete inhibition of the
thrombin-induced activation of
PLA2 (Fig. 8), further suggesting that thrombin stimulation results in an activation of endothelial cell
Ca2+-independent
PLA2.

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Fig. 8.
Effect of pretreatment of endothelial cells with bromoenol lactone
(BEL; 10 µM, 10 min) on basal and thrombin-stimulated (0.05 U/ml, 1 min) PLA2 activity in endothelial
cells. PLA2 activity was measured
by incubating 75 µg endothelial cell protein with 100 µmol/l
(16:0,[3H]18:1)
plasmenylcholine,
(16:0,[3H]18:1)
alkylacyl glycerophosphocholine, and
(16:0,[3H]18:1)
phosphatidylcholine in absence of
Ca2+ (4 mM EGTA) at 37°C for 5 min. Values are means ± SE of independent results derived from
endothelial cells isolated from 6 animals.
* P < 0.05, ** P < 0.01 compared with
control (untreated) values.
+ P < 0.05 compared with corresponding thrombin-stimulated values.
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BEL (10 µM, 10 min) had no significant effect on basal levels of
LPlasC or LPC in endothelial cells but completely blocked the
thrombin-stimulated increase in LPlasC content (Fig.
9). Pretreatment of the endothelial cell
monolayer with BEL (10 µM, 10 min) completely blocked the
thrombin-induced increase in arachidonic acid release. Release from
pretreated cells stimulated with thrombin was significantly less at
each stimulation time compared with control, untreated endothelial
cells (Fig. 10). Pretreatment of
endothelial cells with MAFP (5 µM, 15 min) had no effect on
thrombin-stimulated PLA2 activity,
LPlasC production, or arachidonic acid release (data not shown).
Because MAFP has been demonstrated to inhibit both
Ca2+-dependent and
Ca2+-independent cytosolic
PLA2 isoforms (2), it does not
appear from our results that cytosolic
PLA2 has a role in
thrombin-stimulated endothelial cells. Thus thrombin stimulation of
endothelial cells leads to a rapid increase in LPlasC and arachidonic
acid release that is mediated by the action of
Ca2+-independent
PLA2 hydrolysis of membrane
plasmalogen phospholipids.

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Fig. 9.
Effect of pretreatment of endothelial cells with BEL (10 µM, 10 min)
on basal and thrombin-stimulated (0.05 U/ml, 1 min) LPlasC and LPC
content. BEL had no effect on basal LPlasC but completely inhibited
thrombin-stimulated increases in LPlasC content. No change in LPC
content was observed with BEL or thrombin. Values are means ± SE of
independent results from endothelial cells isolated from 6 animals.
** P < 0.01 compared with
corresponding control values.
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Fig. 10.
Time course for
[3H]arachidonic acid
release from endothelial cells following stimulation with thrombin
(0.05 U/ml; ). Pretreatment with BEL (10 µM, 10 min) completely
blocked thrombin-stimulated
[3H]arachidonic acid
release ( ). Dotted line,
[3H]arachidonic acid
release in untreated cells over same time course. Values are means + SE
of independent results derived from endothelial cells isolated from 4 animals. * P < 0.05, ** P < 0.01 compared with
release in untreated cells.
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Pretreatment of endothelial cells with either dibucaine (50 µM, 30 min) or mepacrine (10 µM, 30 min) had no effect on either basal or
thrombin-stimulated PLA2 activity
(data not shown). These inhibitors have been used historically as
phospholipase inhibitors, since they are reported to interfere with the
substrate-enzyme interface and physically prevent hydrolysis of the
substrate (22). However, endothelial cell
Ca2+-independent
PLA2 was found to be resistant to
these classic inhibitors.
The tumor-promoting phorbol ester PMA can activate protein kinase C
(PKC) during acute exposure or downregulate PKC following chronic
exposure (16, 29). Endothelial cells were incubated with 1 µM PMA for
5 min (activation of PKC) and 24 h (downregulation of PKC) before
stimulation with thrombin (0.05 U/ml, 1 min). Exposure of endothelial
cells to PMA for 5 min resulted in a significant increase in
PLA2 activity measured using
(16:0,[3H]18:1)
plasmenylcholine (Fig.
11A)
and (16:0,[3H]18:1)
alkylacyl glycerophosphocholine substrates (Fig.
11B). No effect on
PLA2 activity measured with
(16:0,[3H]18:1)
phosphatidylcholine substrate was observed following 5 min PMA exposure
(data not shown). Subsequent stimulation with thrombin following acute
exposure to PMA resulted in a further increase in
PLA2 activity measured using
plasmenylcholine or alkylacyl glycerophosphocholine substrates (Fig.
11). The increase in PLA2 activity
produced by PMA and thrombin in combination was greater than that
observed for either compound alone. In contrast, exposure of
endothelial cells to PMA for 24 h resulted in a significant decrease in
PLA2 activity measured using all
substrates tested (Fig. 11). Thrombin-stimulated endothelial cells that
had been exposed previously to PMA for 24 h demonstrated no increase in PLA2 activity (Fig. 11). These
results support our previously published findings (8) that activation
of PKC appears to mediate thrombin-stimulated increases in
PLA2 activity in endothelial
cells.

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Fig. 11.
Modulation of basal and thrombin-stimulated
PLA2 activity in endothelial cells
pretreated with 1 µM phorbol 12-myristate 13-acetate (PMA) for 5 min
[activates protein kinase C (PKC)] or 24 h (downregulates
PKC). PLA2 activity was measured
using
(16:0, [3H]18:1)
plasmenylcholine (A) or
(16:0,[3H]18:1)
alkylacyl glycerophosphocholine (B).
Activity measured with
(16:0,[3H]18:1)
phosphatidylcholine is not shown (see text for details). Values are
means ± SE of independent results derived from endothelial cells
isolated from 4 animals. * P < 0.05, ** P < 0.01 between
control and thrombin-stimulated values within same pretreatment group.
+ P < 0.05 between basal activity in PMA-treated group compared with
basal activity in untreated cells.
|
|
Activation of PKC with PMA for 5 min increased LPlasC in endothelial
cells (Fig. 12). Thrombin stimulation of
endothelial cells following activation of PKC did not result in a
further increase in LPlasC content (Fig. 12). Downregulation
of PKC by 24 h of exposure to PMA completely blocked the
thrombin-stimulated increase in LPlasC. No change in LPC content in
endothelial cells was observed with either activation or downregulation
of PKC.

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|
Fig. 12.
Modulation of basal and thrombin-stimulated LPlasC and LPC content in
endothelial cells pretreated with 1 µM PMA for 5 min (activates PKC)
or 24 h (downregulates PKC). Values are means ± SE of independent
results derived from endothelial cells isolated from 4 animals.
** P < 0.01 compared with
control values.
++ P < 0.05 between thrombin-stimulated LPlasC in PMA-treated group
compared with that in untreated cells.
|
|
Taken together, these results indicate that thrombin stimulation of
endothelial cells activates a membrane-associated,
Ca2+-independent
PLA2 that selectively hydrolyzes
plasmalogen phospholipids, resulting in release of LPlasC and
arachidonic acid into the surrounding medium. The selective increase in
LPlasC in endothelial cells is particularly remarkable, since only 13%
of choline phospholipids contain a vinyl ether linkage at the
sn-1 position (Table
2). Because thrombin
stimulation of endothelial cells results in increased PLA2 activity measured using
alkylacyl glycerophosphocholine, it is likely that increased metabolism
of these phospholipids in response to thrombin stimulation would lead
to increased LPAF in endothelial cells. However, the percentage of
alkylacyl glycerophosphocholine in endothelial cells is extremely low,
and we were not able to measure any LPAF content in endothelial cells
under basal conditions or following thrombin stimulation using mass
measurements.
 |
DISCUSSION |
Lethal arrhythmias are precipitated by acute myocardial infarction
related to coronary thrombosis, with a peak incidence immediately following the onset of occlusion and a precipitous decline thereafter. Formation of an intracoronary thrombosis has been linked directly to
the incidence of arrhythmias (8), suggesting that some component of the
clot is arrhythmogenic. We previously demonstrated an increase in total
choline lysophospholipid release (LPlasC and LPC measured together)
from aortic endothelial cells following thrombin stimulation (16) that
contributes to the increase in lysophospholipid content measured in
venular effluents from an ischemic area. We have now demonstrated that
increased lysophospholipid production is the direct result of
activation of a membrane-associated,
Ca2+-independent
PLA2 in endothelial cells. The
stimulation of PLA2 occurs at
concentrations of thrombin that are known to elicit a variety of other
responses (9, 10, 13) and at concentrations considerably lower than
those measured previously in the vicinity of evolving thrombi (16).
The majority of PLA2 activity in
endothelial cells was found to be membrane associated, with that
measured in the cytosol being an order of magnitude smaller. In
addition, endothelial cell PLA2
was found to be Ca2+-independent,
since the addition of Ca2+ to the
assay buffer had no effect on PLA2
activity compared with that in the absence of
Ca2+. Endothelial cell
PLA2 also displays a preference
for phospholipid substrates with an ether covalent linkage at the
sn-1 position and arachidonate at the
sn-2 position. Thrombin stimulation of endothelial cells resulted in increased
PLA2 activity measured using
substrates with either a vinyl ether or alkyl ether linkage at the
sn-1 position. Along with the increase
in PLA2 activity, we measured an
increase in arachidonic acid release and LPlasC production in response
to thrombin stimulation. These results suggest the selective turnover
of plasmalogen phospholipids in endothelial cell membranes in response
to thrombin stimulation. This is particularly important, since in
endothelial cells only 13% of choline glycerophospholipids are
plasmalogens. However, 40% of arachidonate esterified to choline
phospholipids is found in plasmalogens; in fact, 85% of choline
plasmalogens contain arachidonate at the
sn-2 position (18). Thus, because it
appears that arachidonic acid release in response to thrombin
stimulation of endothelial cells is derived from plasmenylcholine, this
particular group of phospholipids represents a highly metabolically
active pool.
Our findings that arachidonic acid production in endothelial cells is
dependent on the activity of a
Ca2+-independent
PLA2 that is selective for
ether-linked phospholipids are supported by a previous study that
determined that eicosanoid production in bovine aortic endothelial
cells (BAEC) is also dependent on a
Ca2+-independent,
plasmalogen-selective PLA2 (24).
The authors determined that arachidonate turnover in BAEC was decreased
after 15 min of hypoxia and continued to decrease for up to 1 h of
hypoxia. Thus there is evidence to suggest that, after the initial
increase in PLA2 activity
occurring in response to thrombin stimulation soon after the onset of
myocardial ischemia, there may be a later decrease in
PLA2 activity associated with
longer ischemic intervals. This phenomenon could be further studied by
combining thrombin stimulation with hypoxic conditions and measuring
changes in PLA2 activity.
The ability of the agonist peptide SFLL to increase
Ca2+-independent
PLA2 activity and LPlasC
production in endothelial cells suggests that
PLA2 is coupled to the G
protein-coupled receptor that was originally cloned from a human
megakaryoblastic cell line and is a seven-transmembrane domain receptor
that is activated by proteolytic cleavage of the amino terminus by
thrombin. Although other thrombin receptors may also exist, the
evidence that the proteolytically cleaved receptor is expressed on
human endothelial cells includes the presence of mRNA encoding the
receptor, several antibody binding studies, and the ability of SFLL to
mimic the effects of thrombin on endothelial cells (12, 13, 21, 28).
Our results indicate that PKC regulates endothelial cell
PLA2, since activation of PKC by
brief exposure to PMA enhanced basal and thrombin-stimulated
PLA2 and loss of PKC activity by
prolonged exposure to PMA blocked thrombin-stimulated
PLA2 activity and significantly
decreased basal PLA2 activity.
These results support our previous study demonstrating that choline
lysophospholipid release was dependent on PKC activity (16).
Inhibition of thrombin-induced
PLA2 activity, arachidonic acid
release, and LPlasC production by the selective
Ca2+-independent
PLA2 inhibitor BEL demonstrates
that thrombin stimulation of PLA2
in endothelial cells is mediated through a
Ca2+-independent enzyme. Recently,
BEL has been demonstrated to inhibit phosphatidic phosphohydrolase
(PAP) with a IC50 of 8 µM (2). However, we observed complete inhibition of LPlasC and arachidonic acid
release with 10 µM BEL, whereas complete inhibition of PAP has not
been observed with concentrations of BEL >50 µM. However, in this
study, we have also demonstrated that activation of
PLA2 is dependent on PKC activity,
and it is possible that sequential action of phospholipase D and PAP
leading to diacylglycerol production and activation of PKC may be a
pathway through which BEL inhibits PLA2 activity.
Activation of endothelial cell
PLA2, accompanied by increased
production of arachidonic acid and lysophospholipids, has important implications in the setting of ischemic heart disease. Both arachidonic acid and lysophospholipids may affect membrane function directly (20)
or serve as precursors for biochemically active metabolites: lysophospholipids can serve as platelet-activating factor precursors, whereas arachidonic acid serves as the precursor for eicosanoid production (4). Previous studies in both animals and humans showed an
increase in LPC concentration in venular and lymphatic effluents from
ischemic areas (1, 25, 26). Extramyocytic LPC may become incorporated
into the outer leaflet of the ischemic myocyte sarcolemma, where its
catabolism is inhibited by accumulation of long-chain acylcarnitine
(for review, see Ref. 20). Extracellular LPC has been demonstrated to
have profound effects on the electrophysiological properties of
normoxic myocytes and thus can directly influence the production of
arrhythmias in the ischemic heart. In addition, LPlasC is capable of
producing changes in the action potential parameters of cardiac
myocytes at much lower concentrations than those reported previously
for LPC (19). Thus the finding that thrombin stimulation of endothelial
cells results in the selective production of LPlasC has even more
implications than we first thought from our previous studies (16).
In conclusion, we have now demonstrated that thrombin stimulation of
porcine aortic endothelial cells results in activation of a
membrane-associated,
Ca2+-independent
PLA2 that is selective for
ether-linked phospholipids. Activation of this
PLA2 leads to an increase in
arachidonic acid release and accumulation of LPlasC, both of which may
have important implications in the setting of arrhythmogenesis
associated with myocardial ischemia.
We thank RaeTreal McCrory for technical assistance and Monsanto
Corporate Research (St. Louis, MO) for the gift of SFLL and FSLL peptides.
This research was supported in part by the American Heart Association,
Missouri Affiliate (J. McHowat) and National Center (J. McHowat and M. H. Creer), and by National Institutes of Health Grant HL-54907-01 (J. McHowat).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: J. McHowat, Department of Pathology, St.
Louis University School of Medicine, 1402 S. Grand Ave., St. Louis, MO
63104.