Selective hydrolysis of plasmalogens in endothelial cells following thrombin stimulation

Michael H. Creer and Jane McHowat

Department of Pathology, St. Louis University School of Medicine, St. Louis, Missouri 63104

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

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.

lysoplasmenylcholine; pig; bromoenol lactone; arachidonic acid

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   PLA2 activity in subcellular fractions from porcine aortic endothelial cells

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).


View larger version (19K):
[in this window]
[in a new window]
 
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 (bullet ), (16:0,[3H]18:1) alkylacyl glycerophosphocholine (open circle ), 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.

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.


View larger version (22K):
[in this window]
[in a new window]
 
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.

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).


View larger version (20K):
[in this window]
[in a new window]
 
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 (bullet ), (16:0, [3H]18:1) alkylacyl glycerophosphocholine (open circle ), 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.


View larger version (22K):
[in this window]
[in a new window]
 
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.

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).


View larger version (22K):
[in this window]
[in a new window]
 
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 (bullet ), (16:0,[3H]18:1) alkylacyl glycerophosphocholine (open circle ), 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.


View larger version (21K):
[in this window]
[in a new window]
 
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.

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.


View larger version (28K):
[in this window]
[in a new window]
 
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.

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.


View larger version (23K):
[in this window]
[in a new window]
 
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.

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.


View larger version (22K):
[in this window]
[in a new window]
 
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.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 10.   Time course for [3H]arachidonic acid release from endothelial cells following stimulation with thrombin (0.05 U/ml; bullet ). Pretreatment with BEL (10 µM, 10 min) completely blocked thrombin-stimulated [3H]arachidonic acid release (triangle ). 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.

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.


View larger version (17K):
[in this window]
[in a new window]
 


View larger version (15K):
[in this window]
[in a new window]
 
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.


View larger version (25K):
[in this window]
[in a new window]
 
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.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Phospholipid composition of isolated porcine endothelial cells

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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.

    ACKNOWLEDGEMENTS

We thank RaeTreal McCrory for technical assistance and Monsanto Corporate Research (St. Louis, MO) for the gift of SFLL and FSLL peptides.

    FOOTNOTES

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.

Received 19 June 1998; accepted in final form 20 August 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Akita, H., M. H. Creer, K. A. Yamada, B. E. Sobel, and P. B. Corr. Electrophysiologic effects of intracellular lysophosphoglycerides and their accumulation in cardiac lymph with myocardial ischemia in dogs. J. Clin. Invest. 78: 271-280, 1986[Medline].

2.   Balsinde, J., and E. A. Dennis. Bromoenol lactone inhibits magnesium-dependent phosphatidate phosphohydrolase and blocks triacylglycerol biosynthesis in mouse P388D1 macrophages. J. Biol. Chem. 271: 31937-31941, 1996[Abstract/Free Full Text].

3.   Bligh, E. G., and W. J. Dyer. A rapid method of total lipid extraction and purification. Can. J. Physiol. 37: 911-917, 1959.

4.   Chang, J., J. H. Musser, and H. McGregor. Phospholipase A2: function and pharmacological regulation. Biochem. Pharmacol. 36: 2429-2436, 1987[Medline].

5.   Creer, M. H., and R. W. Gross. Reversed-phase high-performance liquid chromatographic separation of molecular species of alkyl ether, vinyl ether, and monoacyl lysophospholipids. J. Chromatogr. 338: 61-69, 1985[Medline].

6.   Davies, M. J., and A. Thomas. Thrombosis and acute coronary artery lesions in sudden cardiac ischemic death. N. Engl. J. Med. 310: 1137-1140, 1984[Abstract].

7.   Dobmeyer, D. J., P. B. Corr, and M. H. Creer. A sensitive, radiometric assay for lysophosphatidylcholine. Anal. Biochem. 185: 36-43, 1990[Medline].

8.   Goldstein, J. A., M. C. Butterfield, Y. Onishi, T. J. Shelton, and P. B. Corr. Arrhythmogenic influence of intracoronary thrombosis during acute myocardial ischemia. Circulation 90: 139-147, 1994[Abstract].

9.   Graham, D. J., and J. A. Alexander. The effects of thrombin on bovine aortic endothelial and smooth muscle cells. J. Vasc. Surg. 11: 307-313, 1990[Medline].

10.   Halldorsson, H., M. Kjeld, and G. Thorgeirsson. Role of phosphoinositides in the regulation of endothelial protacyclin production. Arteriosclerosis 8: 147-154, 1988[Abstract].

11.   Hazen, S. L., L. A. Zupan, R. H. Weiss, D. P. Getman, and R. W. Gross. Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2: mechanism-based discrimination between calcium-dependent and -independent phospholipases A2. J. Biol. Chem. 266: 7227-7232, 1991[Abstract/Free Full Text].

12.   Hein, L., K. Ishii, S. R. Coughlin, and B. K. Kobilka. Intracellular targeting and trafficking of thrombin receptors. A novel mechanism for resensitization of a G protein-coupled receptor. J. Biol. Chem. 269: 27719-27726, 1994[Abstract/Free Full Text].

13.   Horvat, R., and G. E. Palade. The functional thrombin receptor is associated with the plasmalemma and a large endosomal network in cultured human umbilical vein endothelial cells. J. Cell Sci. 108: 1155-1164, 1995[Abstract/Free Full Text].

14.   Man, R. Y. K., A. A. A. Kinnaird, I. Bihler, and P. C. Choy. The association of lysophosphatidylcholine with isolated cardiac myocytes. Lipids 25: 450-454, 1990[Medline].

15.   Markwell, M. A. K., S. M. Haas, N. E. Tolbert, and L. L. Bieber. Protein determination in membrane and lipoprotein samples: manual and automated procedures. Methods Enzymol. 72: 296-303, 1981[Medline].

16.   McHowat, J., and P. B. Corr. Thrombin-induced release of lysophosphatidylcholine from endothelial cells. J. Biol. Chem. 268: 15605-15610, 1993[Abstract/Free Full Text].

17.   McHowat, J., and M. H. Creer. Lysophosphatidylcholine accumulation in cardiomyocytes requires thrombin activation of Ca2+-independent PLA2. Am. J. Physiol. 272 (Heart Circ. Physiol. 41): H1972-H1980, 1997[Abstract/Free Full Text].

18.   McHowat, J., J. H. Jones, and M. H. Creer. Gradient elution reverse-phase chromatographic isolation of individual glycerophospholipid molecular species. J. Chromatogr. B Biomed. Appl. 702: 21-32, 1997.

19.   McHowat, J., S. Liu, and M. H. Creer. Selective hydrolysis of plasmalogen phospholipids by Ca2+-independent PLA2 in hypoxic ventricular myocytes. Am. J. Physiol. 274 (Cell Physiol. 43): C1727-C1737, 1998[Abstract/Free Full Text].

20.   McHowat, J., K. A. Yamada, J. Wu, G.-X. Yan, and P. B. Corr. Recent insights pertaining to sarcolemmal phospholipid alterations underlying arrhythmogenesis in the ischemic heart. J. Cardiovasc. Electrophysiol. 4: 288-310, 1993[Medline].

21.   Mirza, H., V. Yatsula, and W. F. Bahou. The proteinase activated receptor-2 (PAR-2) mediates mitogenic responses in human vascular endothelial cells. J. Clin. Invest. 97: 1705-1714, 1996[Abstract/Free Full Text].

22.   Mukherjee, A. B., L. Miele, and N. Pattabiraman. Phospholipase A2 enzymes: regulation and physiological role. Biochem. Pharmacol. 48: 1-10, 1994[Medline].

23.   Naski, M. C., and J. A. Shafer. A kinetic model for the alpha -thrombin-catalyzed conversion of plasma levels of fibrinogen to fibrin in the presence of antithrombin III. J. Biol. Chem. 266: 13003-13010, 1991[Abstract/Free Full Text].

24.   Patton, G. M., H. Kadowaki, H. Albadawi, H. M. Soler, and M. T. Watkins. Effect of hypoxia on steady-state arachidonic acid metabolism in bovine aortic endothelial cells. Am. J. Physiol. 272 (Heart Circ. Physiol. 41): H1426-H1436, 1997[Abstract/Free Full Text].

25.   Sedlis, S. P., J. M. Sequeira, and H. M. Altszuler. Coronary sinus lysophosphatidylcholine accumulation during rapid atrial pacing. Am. J. Cardiol. 66: 695-698, 1990[Medline].

26.   Snyder, D. W., W. A. Crafford, Jr., J. L. Glashow, D. Rankin, B. E. Sobel, and P. B. Corr. Lysophosphoglycerides in ischemic myocardium effluents and potentiation of their arrhythmogenic effects. Am. J. Physiol. 231 (Heart Circ. Physiol. 12): H700-H707, 1981.

27.   Vu, T.-K., D. T. Hung, V. I. Wheaton, and S. R. Coughlin. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64: 1057-1068, 1991[Medline].

28.   Woolkalis, M. J., T. M. DeMelfi, Jr., N. Blanchard, J. A. Hoxie, and L. F. Brass. Regulation of thrombin receptors on human umbilical vein endothelial cells. J. Biol. Chem. 270: 9868-9875, 1995[Abstract/Free Full Text].

29.   Xing, M., and P. A. Insel. Protein kinase C-dependent activation of cytosolic phospholipase A2 and mitogen-activated protein kinase by alpha1-adrenergic receptors in Madin-Darby canine kidney cells. J. Clin. Invest. 97: 1302-1310, 1996[Abstract/Free Full Text].


Am J Physiol Cell Physiol 275(6):C1498-C1507
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society