Department of Pathology, St. Louis University Medical School, St. Louis, Missouri 63104
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ABSTRACT |
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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
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INTRODUCTION |
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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).
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MATERIALS AND METHODS |
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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--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(-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.
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RESULTS |
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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.
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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).
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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
protein1 · 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).
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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.
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DISCUSSION |
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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).
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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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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.
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