Thrombin activates a membrane-associated calcium-independent PLA2 in ventricular myocytes

Jane McHowat and Michael H. Creer

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

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Activation of phospholipase A2 (PLA2) and accumulation of lysophosphatidylcholine contribute importantly to arrhythmogenesis during acute myocardial ischemia. We examined thrombin stimulation of PLA2 activity in isolated ventricular myocytes. Basal and thrombin-stimulated cardiac myocyte PLA2 activity demonstrated a distinct preference for sn-1 ether-linked phospholipids with arachidonate esterified at the sn-2 position. The majority of PLA2 activity was calcium independent and membrane associated. Thrombin stimulation of membrane-associated PLA2 occurs in a time- and concentration-dependent fashion. An increase in PLA2 activity was also observed using the synthetic peptide SFLLRNPNDKYEPF (the tethered ligand generated by thrombin cleavage of its receptor). Bromoenol lactone, a selective inhibitor of calcium-independent PLA2, completely blocked thrombin-stimulated increases in PLA2 activity and arachidonic acid release. No significant inhibition of thrombin-induced PLA2 was observed following pretreatment with mepacrine or dibucaine. These data confirm the presence of high-affinity thrombin receptors on isolated cardiac myocytes and demonstrate the specific activation of a unique membrane-associated, calcium-independent PLA2 following thrombin receptor ligation.

arachidonic acid release; bromoenol lactone; lysophosphatidylcholine; rabbit; phospholipase A2

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

SUDDEN CARDIAC DEATH IN patients with ischemic heart disease results primarily from malignant ventricular arrhythmias following acute occlusive intracoronary thrombus formation superimposed on an ulcerated atherosclerotic plaque (5). It has been demonstrated that thrombotic coronary occlusion results in a much greater incidence of malignant ventricular arrhythmias than does nonthrombotic balloon occlusion (8), indicating that products released from or associated with an intracoronary thrombus may directly or indirectly influence the electrophysiological properties of ischemic myocytes. Although multiple factors derived from the thrombus or induced during thrombotic occlusion could be responsible, thrombin is likely to play an important role. This conclusion is based on the fact that thrombin is the final effector of activation of the coagulation system, is a potent receptor agonist, and is generated in sufficiently high concentrations (19, 25) to result in stimulation of virtually any thrombin receptor-bearing cell in the vicinity of the thrombus, particularly in the ischemic region distal to the site of thrombotic occlusion where endothelial cell injury leads to increased vascular permeability and release of thrombin into the extravascular space. Thrombin receptor-mediated signaling has been shown to regulate a diverse number of metabolic processes, including activation of phospholipases in platelets (14), endothelial cells (16, 25), and cardiac myocytes (17, 20, 21).

Among the metabolic alterations elicited by ischemia, the accumulation of amphiphilic lipid metabolites [particularly lysophosphatidylcholine (LPC; this term is also used to collectively refer to 1-O-alkyl, 1-O-alk-1'-enyl, and 1-O-acyl forms of monoradyl choline glycerophospholipids)] has been shown to contribute importantly to the development of electrophysiological dysfunction (18). The accumulation of LPC during ischemia is the result of the rapid activation of myocardial phospholipase A2 (PLA2) accompanied by net inhibition of LPC catabolism (7, 17, 18). We have shown that thrombin can stimulate the production of LPC in vascular endothelial cells and isolated ventricular myocytes (16, 17, 20).

The present study was undertaken to characterize basal and thrombin-stimulated PLA2 in isolated rabbit ventricular myocytes and to determine whether any observed effects of thrombin were the result of specific activation of surface membrane thrombin receptors. We demonstrated that thrombin stimulation of isolated adult rabbit ventricular myocytes activates membrane-associated, calcium-independent PLA2, accompanied by an increase in arachidonic acid release. Activation of PLA2 was mediated via specific interaction of thrombin with its receptor and was blocked by the calcium-independent PLA2 inhibitor (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL).

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Most reagents were purchased from Sigma Chemical (St. Louis, MO). The stock solutions of thrombin (Sigma), SFLLRNPNDKYEPF (SFLL), or FSLLRNPNDKYEPF (FSLL) (both gifts from Monsanto Corporate Research, St. Louis, MO) were made in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer. [3H]oleic acid and [3H]arachidonic acid were purchased from DuPont NEN. L-alpha -Phosphatidylcholine from bovine heart was purchased from Avanti Polar Lipids. BEL was a gift from Hoffmann-La Roche (Nutley, NJ).

Isolation of ventricular myocytes. Adult female rabbits weighing 2-3 kg were anesthetized with intravenous pentobarbital sodium (50 mg/kg), and the hearts were rapidly removed. Hearts were mounted on a Langendorff perfusion apparatus and perfused for 5.5 min with a Tyrode solution containing (in mmol/l) 118 NaCl, 4.8 KCl, 1.2 CaCl2, 1.2 MgCl2, 24 NaHCO3, 1.2 KH2PO4, and 11 glucose; the Tyrode solution was saturated with 95% O2-5% CO2 to yield a pH of 7.4. This was followed by a 3.5-min perfusion with a calcium-free Tyrode solution containing ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; 100 µM) and a final perfusion for 20 min with the Tyrode solution containing 100 µM calcium and 0.033% collagenase (type II, Worthington Biochemical). The left and right ventricles were cut into small pieces and placed in two Erlenmeyer flasks containing fresh enzyme solution; flasks were then shaken in a Dubnoff metabolic shaker at 37°C for 15 min, with 95% O2-5% CO2 blowing into each flask. The first harvest of myocytes was discarded. Cells from the next three harvests were combined and washed with a HEPES buffer containing (in mmol/l) 133.5 NaCl, 4.8 KCl, 1.2 MgCl2, 1.2 KH2PO4, 10 HEPES, and 10 glucose, plus 300 µM CaCl2, pH adjusted to 7.4 with 10 N NaOH. Extracellular calcium concentration was increased to 1.2 mM in three stages at intervals of 20 min. Elongated myocytes were separated from rounded nonviable cells by repeated differential sedimentation.

PLA2 assays. At the end of the stimulation period, the myocyte suspension was immediately placed on ice and sonicated for 10 s. After initial sonication, 2 mM dithiothreitol (DTT) and 10% glycerol were added to the cell suspension. The suspension was sonicated on ice a further three times for 10 s, and the sonicate was centrifuged at 14,000 g for 10 min. The resultant supernatant fraction was centrifuged at 100,000 g for 60 min to separate the membrane fraction (pellet) from the cytosolic fraction (supernatant). The membranes were resuspended in buffer containing (in mmol/l) 250 sucrose, 10 KCl, 10 imidazole, 5 EDTA, and 2 DTT with 10% glycerol, pH 7.8 with 10 N KOH. PLA2 activity in subcellular fractions was assessed by incubating enzyme (8 µg membrane protein or 200 µg cytosolic protein) with 100 µM sn-2 radiolabeled plasmenylcholine, phosphatidylcholine, or alkylacyl glycerophosphorylcholine in assay buffer containing 100 mM tris(hydroxymethyl)aminomethane and 10% glycerol (pH 7.0) with either 4 mM EGTA or 10 mM calcium at 37°C for 5 min in a total volume of 200 µl. The reaction was initiated by adding the substrate as a concentrated stock solution in ethanol (5 µl total volume), which was injected into a total volume of 200 µl aqueous buffer to achieve a final substrate concentration of 100 µM. Reactions were terminated by the addition of 100 µl butanol; then tubes were vortexed and centrifuged at 2,000 g for 5 min. Released radiolabeled fatty acid was isolated by application of 25 µl of the butanol phase to channeled silica gel G plates, development in petroleum ether-diethyl ether-acetic acid (70:30:1, vol/vol), and subsequent quantification by liquid scintillation spectrometry. The reaction conditions selected resulted in linear reaction velocities with respect to both time and total protein concentration for each substrate examined. Protein content of each sample was determined by the Lowry method utilizing freeze-dried bovine serum albumin (Bio-Rad Laboratories) as the protein standard as described previously (15).

Under our PLA2 assay conditions, we found linear reaction velocities with respect to protein concentrations for 2-20 µg membrane protein and 100-500 µg cytosolic protein. Incubation times were limited to 5 min or less as longer incubation times were associated with a nonlinear increase in enzyme activity. Maximal reaction velocities were consistently achieved with substrate concentrations >50 µM. We used 100 µM as the substrate concentration for our assays to ensure that maximum rate measurements were being made and to minimize any effects of isotope dilution by endogenous substrates (in all assays, we calculated that exogenous radiolabeled substrate was present in >10-fold molar excess over all potential endogenous substrates in both cytosolic and membrane fractions). With the use of both one- and two-dimensional thin-layer chromatography (TLC) separation techniques, free fatty acid was the only radiolabeled product produced following incubation of substrate with cytosol and membrane protein. The production of labeled fatty acid accounted for >95% of the total decrease in radiolabeled substrate. Thus, on the basis of the stoichiometric production of labeled fatty acid and decrease in sn-2 radiolabeled diradyl choline phospholipid substrate and absence of other radiolabeled products, we can confidently equate the rate of fatty acid production with PLA2 activity. These results do not rule out the presence of other phospholipases but clearly indicate that PLA2 is the predominant phospholipase measured under our assay conditions.

Synthesis of phospholipid substrates. Lysoplasmenylcholine was prepared by alkaline hydrolysis of bovine heart choline glycerophospholipid as described previously (3). Radiolabeled plasmenylcholine (1-O-hexadec-1'-enyl-2-acyl-sn-glycero-3-phosphocholine) was prepared by reacting the unlabeled 16:0 lysoplasmenylcholine with radiolabeled fatty anhydride utilizing N,N-dimethyl-4-aminopyridine as a catalyst (10). Radiolabeled fatty anhydride was prepared from [9,10-3H]oleic acid or [5,6,8,9,11,12,14,15-3H]arachidonic acid utilizing dicyclohexylcarbodiimide-mediated condensation of the fatty acid. Radiolabeled plasmenylcholine was purified by passing the reaction mixture through an amine solid-phase extraction column followed by normal phase high-performance liquid chromatography (HPLC) using a Partisil SCX column (9). Synthesis and purification of sn-2 radiolabeled phosphatidylcholine and alkyl ether choline glycerophospholipid molecular species were performed similarly, utilizing the appropriate radiolabeled fatty acid and palmitoyl LPC or 1-O-hexadecyl-sn-glycero-3-phosphocholine (lyso-platelet-activating factor). The final purity of all synthetic products was confirmed by demonstration of comigration of synthetic product and standards using two different TLC systems, normal phase HPLC (9) and reverse-phase HPLC separation of the individual phospholipid molecular species (4). The radiochemical purity of the final products was determined by TLC analysis and liquid scintillation spectrometry of the zones corresponding to fatty acid and diradyl choline glycerophospholipid, and the specific activity of the final products was confirmed by liquid scintillation spectrometry and phosphate assay. The synthetic products were stored in the dark at -20°C and were repurified by normal phase HPLC if >3% of the total radioactivity migrated as free fatty acid following TLC analysis.

Measurement of total arachidonic acid release. Arachidonic acid release was determined by measuring [3H]arachidonic acid released into the surrounding medium from myocyte suspensions labeled previously with [3H]arachidonic acid. Briefly, myocyte suspensions (106 myocytes in 10 ml culture medium) were incubated at 37°C with 3 µCi [3H]arachidonic acid for 18 h. This incubation resulted in >70% incorporation of radioactivity into the myocytes. Eighty-five percent of incorporated radioactivity was recovered from phosphatidylcholine or phosphatidylethanolamine phospholipids. After incubation, myocyte suspensions were washed three times with Tyrode solution containing 3.6% bovine serum albumin to remove unincorporated [3H]arachidonic acid. Myocytes were incubated at 37°C for 15 min before being subjected to experimental conditions. At the end of the stimulation period, myocyte suspensions were centrifuged, and the supernatant was removed. Myocyte pellets were dissolved in 10% sodium dodecyl sulfate, and radioactivity in both supernatant and pellet was quantified by liquid scintillation spectrometry.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

PLA2 activity in isolated rabbit cardiac myocytes. The cytosolic vs. membrane subcellular distribution, calcium requirements, and substrate selectivity of basal and thrombin-stimulated PLA2 activity in isolated rabbit cardiac myocytes are presented in Table 1. Basal cardiac myocyte PLA2 activity exhibited the following three characteristics. 1) The highest specific PLA2 activity was found to be in a membrane-associated form. 2) Basal PLA2 activity was markedly influenced by the nature of the covalent linkage of the sn-1 aliphatic group and the composition of the sn-2 aliphatic group of the substrate molecule. The greatest specific activity was detected with substrates containing a saturated ether (alkyl ether) or vinyl ether (plasmalogen) linkage at the sn-1 position and arachidonate esterified at the sn-2 position. 3) The majority of PLA2 in isolated ventricular myocytes was found to be calcium independent.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Phospholipase A2 activity in isolated rabbit ventricular myocytes

Membrane-associated PLA2 activity measured using plasmalogen substrate was significantly decreased at calcium concentrations >10-6 M (4.75 ± 0.70 to 3.09 ± 0.15 nmol · mg protein-1 · min-1, n = 3). In contrast, increasing the calcium concentration from 10-7 to 10-2 M had little effect on PLA2 activity measured using phosphatidylcholine substrate.

In response to thrombin stimulation (0.05 U/ml, 1 min), there was a significant increase in membrane-associated PLA2 activity with diacyl and plasmalogen substrates. The relative magnitude of the increase in PLA2 activity was the same (~2-fold) for substrates containing either 18:1 or 20:4 fatty acyl groups esterified at the sn-2 position and was observed in both the presence and absence of calcium. No change in cytosolic PLA2 activity was observed in response to thrombin stimulation under any conditions studied.

Because basal and thrombin-stimulated membrane-associated PLA2 activity could not be removed by exposure of the membranes to EGTA, EDTA, submicellar concentrations of detergent, or repeated sonication, the increase in PLA2 activity appears to be the result of activation of latent membrane-localized enzyme activity and not due to translocation of soluble PLA2 activity from the cytosol to the membrane fraction. To further substantiate this conclusion, we separated cytosolic and membrane fractions from isolated ventricular myocytes before thrombin stimulation. Stimulation of the isolated membrane fraction resulted in an increase in PLA2 activity (2.25 ± 0.24 to 7.92 ± 0.35 nmol · mg protein-1 · min-1 using plasmenylcholine and 2.58 ± 0.47 to 8.77 ± 0.24 nmol · mg protein-1 · min-1 using phosphatidylcholine) similar to that observed following thrombin stimulation of intact myocytes. Thus the same magnitude of increase in membrane-associated PLA2 activity was observed even in the complete absence of cytosolic protein, demonstrating that cytosol-to-membrane translocation does not play a significant role in the increase in PLA2 activity observed following thrombin stimulation.

For all subsequent experiments, measurements of PLA2 activity are reported under maximal conditions, i.e., activity using plasmenylcholine substrate measured in the absence of calcium and that with phosphatidylcholine substrate in the presence of calcium. No significant changes in cytosolic PLA2 activity were detected under any experimental conditions; thus only changes in membrane-associated PLA2 activity are discussed.

Thrombin stimulation of isolated cardiac myocytes results in a dose- and time-dependent increase in PLA2 activity. The time course of PLA2 activation in the membrane fraction in response to thrombin (1 U/ml) stimulation is shown in Fig. 1. Membrane-associated PLA2 activity using (16:0, [3H]18:1) plasmenylcholine substrate increased 3.7-fold after a 30-s thrombin stimulation (Fig. 1A). The rapid increase in PLA2 activity was followed by a rapid decline; however, PLA2 activity remained significantly elevated even after 10 min of stimulation (Fig. 1A). Membrane-associated PLA2 activity defined with (16:0, [3H]18:1) phosphatidylcholine substrate increased 2.3-fold after a 1-min thrombin stimulation and rapidly returned to control levels by 2 min (Fig. 1B).


View larger version (10K):
[in this window]
[in a new window]
 


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 1.   Time course of activation of membrane-associated phospholipase A2 (PLA2) activity following 1 U/ml thrombin stimulation using (16:0, [3H]18:1) plasmenylcholine substrate in the presence of 4 mM EGTA (A) and (16:0, [3H]18:1) phosphatidylcholine substrate in the presence of 10 mM Ca2+ (B). Substrates were incubated with 8 µg membrane protein at 37°C for 5 min. * P < 0.05, ** P < 0.01 compared with controls. Values represent means ± SE of independent results derived from 3 separate animals.

Because membrane-associated PLA2 activity with both diacyl and plasmalogen phospholipid substrates was significantly increased after a 1-min thrombin stimulation, additional experiments were performed to define the concentration-response curve for thrombin at this stimulation interval. Membrane-associated PLA2 activity measured using (16:0, [3H]18:1) plasmenylcholine was significantly increased at thrombin concentrations >0.01 U/ml, with a half-maximal effective dose (ED50) of 0.04 ± 0.02 U/ml (Fig. 2A). A maximal response was observed at concentrations over 0.1 U/ml. Membrane-associated PLA2 activity measured using (16:0, [3H]18:1) phosphatidylcholine substrate was significantly increased at concentrations of thrombin over 0.01 U/ml, with maximal stimulation observed with concentrations exceeding 0.1 U/ml (Fig. 2B). The ED50 for phosphatidylcholine-directed PLA2 activity was 0.06 ± 0.02 U/ml.


View larger version (10K):
[in this window]
[in a new window]
 


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2.   Concentration response curve for thrombin-stimulated membrane-associated PLA2 activity using (16:0, [3H]18:1) plasmenylcholine substrate in the presence of 4 mM EGTA (A) and (16:0, [3H]18:1) phosphatidylcholine substrate in the presence of 10 mM Ca2+ (B). Ventricular myocytes were stimulated with thrombin for 1 min. Substrates were incubated with 8 µg membrane protein at 37°C for 5 min. * P < 0.05, ** P < 0.01 compared with control values. Values represent means ± SE of independent results derived from 4 separate animals.

Stimulation of PLA2 by thrombin treatment is dependent on the proteolytic activity of thrombin and results from direct activation of surface membrane thrombin receptors. Hirudin binds to thrombin, rendering thrombin incapable of binding to its receptor and also interferes with the proteolytic function of thrombin (6). Hirudin (0.25 U/ml) added to isolated rabbit cardiac myocytes 10 min before the addition of thrombin (0.05 U/ml, 1 min) completely blocked the increase in PLA2 activity in the membrane fraction defined with either phosphatidylcholine (3.88 ± 0.87 for control vs. 3.84 ± 0.37 nmol · mg protein-1 · min-1 for hirudin + thrombin, n = 6) or plasmenylcholine substrates (4.46 ± 1.10 for control vs. 3.97 ± 0.85 nmol · mg protein-1 · min-1 for hirudin + thrombin, n = 6).

Recent studies have demonstrated that thrombin cleaves its own receptor in the extracellular domain at a specific amino acid located 42 amino acids from the amino terminus of the receptor. This proteolytic cleavage exposes a new amino terminus that functions as a tethered ligand that binds to a specific binding site on the receptor, thereby eliciting receptor activation (2). The 14 amino acids comprising the newly exposed amino terminus form the peptide sequence SFLLRNPNDKYEPF. This SFLL peptide has been shown to specifically stimulate the thrombin receptor directly and to elicit biological responses in platelets, smooth muscle cells, leukocytes, and endothelial cells (20). Stimulation of isolated rabbit myocytes with the 14-amino acid peptide, SFLL, significantly increased PLA2 activity in the membrane fraction in a concentration-dependent manner when either (16:0, [3H]18:1) plasmenylcholine or phosphatidylcholine was used as substrate (Fig. 3). The increase in membrane-associated PLA2 activity with SFLL was almost identical to that elicited by thrombin stimulation. Membrane-associated PLA2 activity defined with plasmenylcholine was significantly increased at concentrations above 0.01 µM and reached levels comparable to the peak thrombin response (a 2.5-fold increase) at 10 µM SFLL (Fig. 3A). Membrane-associated PLA2 activity defined with phosphatidylcholine was also significantly increased at concentrations of SFLL >0.01 µM, with a maximum 2.2-fold increase at 1 µM SFLL (Fig. 3B). At the same concentrations, stimulation of isolated myocytes with FSLL did not result in any activation of PLA2 activity using either substrate (Fig. 3). These data illustrate the specificity of the receptor in that transposition of only the first two amino acids in the peptide sequence completely abolishes the activation of PLA2. The ability of SFLL to stimulate PLA2 activity to the same extent as thrombin is definitive evidence that the response is mediated via a specific thrombin receptor on cardiac myocytes.


View larger version (14K):
[in this window]
[in a new window]
 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3.   Concentration response curve for SFLLRNPNDKYEPF (SFLL)- and FSLLRNPNDKYEPF (FSLL)-stimulated membrane-associated PLA2 activity using (16:0, [3H]18:1) plasmenylcholine substrate in the presence of 4 mM EGTA (A) and (16:0, [3H]18:1) phosphatidylcholine substrate in the presence of 10 mM Ca2+ (B). Ventricular myocytes were stimulated with SFLL or FSLL for 1 min. Substrates were incubated with 8 µg membrane protein at 37°C for 5 min. * P < 0.05, ** P < 0.01 compared with control values. Values represent means ± SE of independent results derived from 3 separate animals.

BEL blocks the increase in membrane-associated PLA2 activity and arachidonic acid release in response to thrombin stimulation. BEL is a potent mechanism-based inhibitor of myocardial calcium-independent PLA2 that is >1,000-fold specific for inhibition of calcium-independent PLA2 in comparison with a variety of calcium-dependent PLA2 (13). Rabbit cardiac myocytes were incubated with 1-10 µM BEL for 10 min before stimulation with thrombin. Basal membrane-associated PLA2 activity was unaffected by BEL at concentrations lower than 5 µM, but at 10 µM BEL basal PLA2 activity defined using (16:0, [3H]18:1) plasmenylcholine substrate was reduced by 38% and basal membrane-associated activity using (16:0, [3H]18:1) phosphatidylcholine was inhibited by 90% (Fig. 4). Pretreatment of isolated rabbit cardiac myocytes with BEL 10 min before thrombin stimulation (0.05 U/ml, 1 min) resulted in significant inhibition of thrombin-stimulated PLA2 activity at concentrations >1 µM (Fig. 4). Thus pretreatment with BEL differentially inhibits basal and thrombin-stimulated PLA2 in a dose-dependent manner. Pretreatment of isolated myocytes with the calcium-dependent PLA2 inhibitors, 10 µM mepacrine or 50 µM dibucaine, for 30 min before thrombin stimulation had no significant effect on basal or thrombin-stimulated PLA2 activity in the membrane using either plasmenylcholine or phosphatidylcholine substrates (data not shown). These data further support our hypothesis that thrombin stimulation of cardiac myocytes selectively activates a calcium-independent PLA2.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of pretreatment of rabbit ventricular myocytes with increasing concentrations of (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL) for 10 min on membrane-associated PLA2 following stimulation with 0.05 U/ml thrombin for 1 min using (16:0, [3H]18:1) plasmenylcholine substrate in the presence of 4 mM EGTA (black-triangle) or (16:0, [3H]18:1) phosphatidylcholine substrate in the presence of 10 mM Ca2+ (bullet ). Effect of BEL pretreatment on basal PLA2 activity using plasmenylcholine (triangle , dotted lines) and phosphatidylcholine (open circle , dotted lines) is also shown. Substrates were incubated with 8 µg membrane protein at 37°C for 5 min. * P < 0.05, ** P < 0.01 compared with corresponding control values. Values represent means ± SE of independent results derived from 3 to 5 separate animals.

Stimulation of isolated ventricular myocytes with thrombin (0.05 U/ml) resulted in a threefold increase in arachidonic acid release after a 1-min stimulation (Fig. 5). Arachidonic acid release remained significantly elevated over control levels for 20 min. Pretreatment with BEL (10 µM, 10 min) had little effect on basal arachidonic acid release (data not shown) but completely blocked the increase in arachidonic acid release observed in response to thrombin stimulation over the 20-min stimulation interval (Fig. 5). These results indicate that thrombin stimulation of calcium-independent PLA2 in isolated ventricular myocytes leads to the cleavage of arachidonic acid from the sn-2 position of membrane phospholipids, which is then released from the cells into the surrounding medium.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of pretreatment of rabbit ventricular myocytes with BEL (10 µM, 10 min) on thrombin-stimulated [3H]arachidonic acid release. Myocytes were stimulated with 0.05 U/ml thrombin for up to 20 min (bullet ). Pretreatment with BEL (×) completely blocked thrombin-stimulated [3H]arachidonic acid release. During the 20-min time period, basal [3H]arachidonic acid release remained constant in the absence of thrombin (dotted line). Values represent means ± SE of independent results derived from 6 separate animals. ** P < 0.01 comparing thrombin stimulation with control values.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this study, we have demonstrated the activation of membrane-associated PLA2 in response to specific agonist stimulation in isolated ventricular myocytes. Ventricular myocyte PLA2 displays a distinct preference for phospholipid substrates containing an ether covalent linkage at the sn-1 position and arachidonate esterified at the sn-2 position and does not require calcium for activity. Previous studies of PLA2 activity prepared by homogenization of whole rabbit myocardium have demonstrated the presence of both cytosolic and membrane-associated enzymes that also exhibit maximal activities in the absence of calcium (i.e., nominally calcium free with mM concentrations of EGTA or EDTA) and demonstrate a preference for arachidonylated ether-linked phospholipid substrates, particularly plasmalogens (7, 10, 11).

Hazen et al. (10) have demonstrated the rapid and reversible activation of a membrane-associated, calcium-independent plasmalogen-selective PLA2 during no-flow ischemia of isolated perfused rabbit hearts in vitro. Variation in the composition of the fatty acyl residue at the sn-2 position and the nature of the covalent linkage of the aliphatic group at the sn-1 position of the substrate molecule influenced maximal PLA2-catalyzed hydrolytic rates in a manner similar to that observed for thrombin-stimulated ventricular myocyte PLA2. The increase in myocardial membrane-associated PLA2 in whole myocardium during ischemia was shown only when plasmalogen phospholipid substrates were used (10). However, more recently, Vesterqvist et al. (22) failed to demonstrate any increase in PLA2 activity during global ischemia in the same model and, in fact, measured a decrease in PLA2 activity following prolonged ischemia. Although Vesterqvist et al. (22) could not demonstrate increased PLA2 activity during ischemia, they did observe increased lysophospholipid content in ischemic myocardium that was not as a result of decreased lysophospholipid catabolism. The reason(s) for the discrepancy in results from these two studies remains unclear but may result from the method used for measuring PLA2 activity or from the fact that Hazen et al. (10) studied PLA2 activity selectively in different subcellular fractions, whereas Vesterqvist et al. (22) measured activity in the whole myocardium. Thus it is possible that the latter authors could be measuring several PLA2 isoforms that may be influenced differentially by ischemia.

In the above studies, the perfused Langendorff heart preparation utilizes buffer perfusion for the isolated heart; thus no blood components are present. In this study, we highlight the importance of one such component as a potential regulator of increased PLA2 activity in myocardial ischemia. We find that membrane-associated PLA2 in cardiac myocytes increases in response to thrombin stimulation when both plasmenylcholine and phosphatidylcholine are utilized as substrates. Accordingly, thrombin stimulation of isolated cardiac myocytes may activate one or more distinct membrane-associated PLA2 with different substrate preferences. This conclusion is supported by the different time course of enzyme activation in response to thrombin stimulation.

Thrombin concentrations as low as 0.05 U/ml result in significant activation of membrane-associated, calcium-independent PLA2 in isolated ventricular myocytes. Thrombin levels adjacent to evolving coronary thrombi have been shown to be as high as 9 U/ml (19, 23); thus we would expect maximal PLA2 activation to occur in ventricular myocytes during myocardial ischemia. Stimulation of PLA2 activity by the thrombin receptor-directed peptide SFLL indicates that membrane-associated PLA2 activation is mediated through specific interaction of thrombin with its receptor. Concentrations of SFLL required to stimulate PLA2 activity to the same extent as thrombin are much higher than the corresponding thrombin concentrations. This may be due to several factors, such as the high affinity of thrombin for its receptor and the close proximity of the tethered ligand to its receptor following thrombin cleavage.

Inhibition of thrombin-induced increases in PLA2 activity and arachidonic acid release by the selective calcium-independent PLA2 inhibitor BEL demonstrate that thrombin stimulation of PLA2 activity in isolated ventricular myocytes is mediated through a calcium-independent enzyme. Recently, BEL has also been shown to inhibit phosphatidic acid phosphohydrolase (PAP) with a 50% inhibitory concentration of 8 µM (1). Because arachidonic acid release may be a reflection of the sequential action of phospholipase D, PAP, and diacylglycerol lipase as described by Balsinde and Dennis (1), inhibition of PAP with BEL can thus result in decreased arachidonic acid release through a mechanism other than its ability to block calcium-independent PLA2. However, in this study, we observed complete inhibition of thrombin-stimulated PLA2 with concentrations as low as 2 µM BEL, whereas complete inhibition of PAP has not been observed even at concentrations of BEL >50 µM (1). We have also demonstrated previously that thrombin stimulation of ventricular myocytes results in increased LPC production (17), which is also completely blocked by 10 µM BEL, even under hypoxic and acidotic conditions that are known to enhance LPC production in response to PLA2 activation (17). Because we observed concomitant increases in PLA2 activity, arachidonic acid release, and LPC production following thrombin stimulation of isolated ventricular myocytes, all of which can be blocked completely by BEL pretreatment at relatively low concentrations (<10 µM), we are confident that the PAP pathway is unlikely to contribute significantly to arachidonic acid release in response to thrombin stimulation.

The results from this study, together with those from our previous study demonstrating an increase in LPC production in isolated ventricular myocytes, provide a potential mechanism for arrhythmogenesis during the early acute phase of myocardial ischemia. Specifically, activation of ventricular myocyte PLA2 contributes to increased production of arachidonic acid and lysophospholipids, including LPC. The accumulation of amphiphilic LPC metabolites during ischemia has been shown to contribute importantly to the development of electrophysiological abnormalities that predispose the onset of ventricular arrhythmias (reviewed in Ref. 18). The LPC metabolites exert their effects by passive incorporation into the surface membrane (sarcolemma) of the cardiac myocyte where they elicit perturbations in the rate and amplitude of random molecular motion of sarcolemmal phospholipids (18). The resultant alterations in phospholipid molecular dynamics modulate the activity of integral membrane proteins such as active transport proteins and ion channels that control ion flux across the sarcolemma and collectively determine the electrophysiological properties of the cell (18). Plasmalogens account for the majority of phospholipid mass in isolated cardiac myocytes (4); thus the activation of cardiac myocyte PLA2 capable of hydrolyzing plasmalogen phospholipids in response to thrombin would result in increased production of lysoplasmenylcholine metabolites. In preliminary studies, we have found that lysoplasmenylcholine metabolites elicit electrophysiological alterations in isolated cardiac myocytes that are qualitatively very similar to those produced by LPC; however, the effects of lysoplasmenylcholine are manifest at a lower concentration (unpublished observations). Accordingly, we hypothesize that thrombin stimulation of cardiac myocytes at sites distal to an occlusive coronary artery thrombus would contribute importantly to the early onset of potentially fatal ventricular arrhythmias as a direct result of PLA2 activation and accumulation of amphiphilic LPC and lysoplasmenylcholine metabolites. This hypothesis is supported by our present findings that demonstrate the rapid (<1 min) activation of membrane-associated cardiac myocyte PLA2 capable of hydrolyzing both diacyl glycerophospholipids and plasmalogens in response to thrombin at concentrations that are considerably lower than those previously reported near an evolving coronary thrombus or in ischemic zones (19) as well as the results of our previous studies demonstrating the rapid accumulation of monoradyl choline glycerophospholipids (collectively designated LPC) in response to thrombin stimulation (20).

    ACKNOWLEDGEMENTS

Research from the authors' laboratory was supported in part by the Department of Veterans Affairs Research Career Development Award Program (to M. H. Creer), Department of Veterans Affairs Merit Review Grant Program (to M. H. Creer), and the American Heart Association, Arkansas Affiliate (to J. McHowat and M. H. Creer).

    FOOTNOTES

Address for reprint requests: J. McHowat, Dept. of Pathology, St. Louis Univ. School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104.

Received 4 August 1997; accepted in final form 4 November 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   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].

2.   Coughlin, S. R. Thrombin receptor structure and function. Thromb. Haemost. 66: 184-187, 1993.

3.   Creer, M. H., and R. W. Gross. Reverse-phase HPLC separation of molecular species of alkyl ether, vinyl ether and monoacyl lysophospholipids. J. Chromatogr. 338: 61-69, 1985[Medline].

4.   Da Torre, S. D., and M. H. Creer. Differential turnover of poyunsaturated fatty acids in plasmalogen and diacyl glycerophospholipids of isolated cardiac myocytes. J. Lipid Res. 32: 1159-1172, 1991[Abstract].

5.   Davies, M. J., and A. Thomas. Thrombosis and acute coronary artery lesions in sudden cardiac ischemic death. N. Engl. J. Med. 310: 1137-1141, 1984[Abstract].

6.   Fenton, J. W., II. Thrombin interactions with hirudin. Semin. Thromb. Hemost. 15: 265-268, 1989[Medline].

7.   Ford, D. A., S. L. Hazen, J. E. Saffitz, and R. W. Gross. The rapid and reversible activation of a calcium-independent plasmalogen-selective phospholipase A2 during myocardial ischemia. J. Clin. Invest. 88: 331-335, 1991[Medline].

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

9.   Gross, R. W., and B. E. Sobel. Isocratic high-performance liquid chromatography separation of phosphoglycerides and lysophosphoglycerides. J. Chromatogr. 197: 79-85, 1980[Medline].

10.   Hazen, S. L., D. A. Ford, and R. W. Gross. Activation of a membrane-associated phospholipase A2 during rabbit myocardial ischemia which is highly selective for plasmalogen substrate. J. Biol. Chem. 266: 5629-5633, 1991[Abstract/Free Full Text].

11.   Hazen, S. L., and R. W. Gross. Idenitification and characterization of human myocardial phospholipase A2 from transplant recipients suffering from end-stage ischemic heart disease. Circ. Res. 70: 486-495, 1992[Abstract].

12.   Hazen, S. L., and R. W. Gross. ATP-dependent regulation of rabbit myocardial cytosolic calcium-independent phospholipase A2. J. Biol. Chem. 266: 14526-14534, 1994[Abstract/Free Full Text].

13.   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].

14.   Kajiyama, Y., T. Murayama, Y. Kitamura, S. Imai, and Y. Nomura. Possible involvement of different GTP-binding proteins in noradrenaline- and thrombin-stimulated release of arachidonic acid in rabbit platelets. Biochem. J. 270: 69-75, 1990[Medline].

15.   Markwell, M. A., 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., 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].

19.   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].

20.   Park, T. H., J. McHowat, R. A. Wolf, and P. B. Corr. Increased lysophosphatidylcholine content induced by thrombin receptor stimulation in adult rabbit cardiac ventricular myocytes. Cardiovasc. Res. 28: 1263-1268, 1994[Medline].

21.   Steinberg, S. F., R. B. Robinson, H. B. Lieberman, D. M. Stern, and M. R. Rosen. Thrombin modulates phosphoinositide metabolism, cytosolic calcium, and impulse initiation in the heart. Circ. Res. 68: 1216-1229, 1991[Abstract].

22.   Vesterqvist, O., C. A. Sargent, G. J. Grover, and M. L. Ogletree. Myocardial calcium-independent phospholipase A2 activity during global ischemia in isolated rabbit hearts. Cardiovasc. Res. 31: 932-940, 1996[Medline].

23.   Wagner, W. R., and J. A. Hubbell. Local thrombin synthesis and fibrin formation in an in vitro thrombosis model result in platelet recruitment and thrombus stabilization on collagen in heparimized blood. J. Lab. Clin. Med. 116: 636-650, 1990[Medline].

24.   Wolf, M. J., and R. W. Gross. The calcium-dependent association and functional coupling of calmodulin with myocardial phospholipase A2. J. Biol. Chem. 271: 20989-20992, 1996[Abstract/Free Full Text].

25.   Zavoico, G. B., J. K. Hrbolich, M. A. Gimbrone, and A. I. Schafer. Enhancement of thrombin- and ionomycin-stimulated prostacyclin and platelet-activating factor production in cultured endothelial cells by a tumor-promoting phorbol ester. J. Cell. Physiol. 143: 596-605, 1990[Medline].


AJP Cell Physiol 274(2):C447-C454
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society