Identification of lysophosphatidylcholine-chlorohydrin in human atherosclerotic lesions.

Lysophosphatidylcholine (LysoPtdCho) levels are elevated in sera in patients with atherosclerosis and in atherosclerotic tissue. Previous studies have shown that reactive chlorinating species attack plasmalogens in human coronary artery endothelial cells (HCAEC), forming lysoPtdCho and lysoPtdCho-chlorohydrin (lysoPtdCho-ClOH). The results herein demonstrate for the first time that lysoPtdCho-ClOH is elevated over 60-fold in human atherosclerotic lesions. In cultured HCAEC, 18:0 lysoPtdCho-ClOH led to a statistically significant increase in P-selectin cell-surface expression, but unlike 18:1 lysoPtdCho did not lead to cyclooxygenase-2 protein expression. These data show that 18:0 lysoPtdCho-ClOH is elevated in atherosclerotic tissue and may have unique pro-atherogenic properties compared to lysoPtdCho.

Prostaglandin production in human coronary artery endothelial cells is modulated differentially by selective phospholipase A(2) inhibitors.

Atherosclerotic plaque formation is a dynamic process involving repeated injury and inflammation of the endothelium. We have demonstrated previously that thrombin and tryptase stimulation of human coronary artery endothelial cells (HCAEC) leads to increased phospholipase A(2) (PLA(2)) activity and generation of membrane phospholipid derived inflammatory metabolites, including eicosanoids and platelet activating factor. Thus, our hypothesis is that selective PLA(2) inhibitors have therapeutic potential as anti-inflammatory agents. Stimulation of confluent HCAEC monolayers with thrombin or tryptase resulted in a concentration and time-dependent increase in both prostaglandin E(2) (PGE(2)) and prostacyclin (PGI(2)) production. Pretreatment with PX-18 to inhibit secretory PLA(2) or BEL to inhibit calcium-independent PLA(2) prior to thrombin or tryptase stimulation resulted in a significant inhibition of both PGI(2) and PGE(2) release. However, pretreatment with methyl arachidonyl fluorophosphonate (MAFP), a widely used inhibitor of cytosolic PLA(2) isoforms, resulted in a significant potentiation of both thrombin and tryptase stimulated PGI(2) and PGE(2) release as a consequence of increased free arachidonic acid production. We conclude that the use of selective PLA(2) inhibitors may be of therapeutic benefit in the development and progression of atherosclerosis, however, the development of such an agent requires rigorous screening.

The therapeutic potential of phospholipase A2 inhibitors in cardiovascular disease.

Leukocyte recruitment and the expression of pro-inflammatory cytokines are prevalent characteristics of early atherogenesis. Recently, several inflammatory mediators have been linked to atheroma formation and inflammatory pathways have been shown to promote thrombosis. The discovery of mast cells, activated T lymphocytes and macrophages in atherosclerotic lesions, the detection of human leukocyte antigen class II expression, and the finding of local secretion of several cytokines all suggest the involvement of immune and inflammatory mechanisms in the pathogenesis of atherosclerosis. Recent research suggests activation of protease activated receptors (PAR) on the surface of endothelial cells may play a role in general mechanisms of inflammation. In previous studies, our laboratory has demonstrated that thrombin (which activates PAR-1) and tryptase (which activates PAR-2) stimulation of endothelial cells results in activation of calcium-independent phospholipase A(2) (iPLA(2)). iPLA(2) plays a critical role in the synthesis of membrane phospholipid-derived inflammatory mediators such as arachidonic acid, platelet activating factor (PAF), and prostaglandins, all demonstrated to be central in both the initiation and propagation of the inflammatory response. Activation of iPLA(2) results in release of choline lysophospholipids from endothelial cells, these metabolites may contribute to the initiation of ventricular arrhythmias following myocardial ischemia as a direct result of incorporation into the myocyte sarcolemma. This biochemical event represents a direct link between occlusion of a coronary vessel and the nearly immediate initiation of arrhythmogenesis often seen in myocardial ischemia.

Phospholipase A2 inhibitors as potential anti-inflammatory agents.

Phospholipase A(2) (PLA(2))-catalyzed hydrolysis of membrane phospholipids results in the stoichiometric production of a free fatty acid, most importantly arachidonic acid, and a lysophospholipid. Both of these phospholipid metabolites serve as precursors for inflammatory mediators such as eicosanoids or platelet-activating factor (PAF). Since it was initially discovered that non-steroidal anti-inflammatory drugs inhibit prostaglandin synthesis, a vast amount of drug development has been performed to selectively inhibit the production of the inflammatory metabolites of arachidonic acid while preserving their protective role. This research has culminated in the development of selective cyclooxygenase-2 (COX-2) inhibitors that act on the inducible, inflammatory COX enzyme, but do not affect the constitutive prostaglandin synthesis in cells that is mediated via COX-1. The development of PLA(2) inhibitors as potential anti-inflammatory agents has also been extensively pursued since the release of arachidonic acid from membrane phospholipids by PLA(3) is one of the rate-limiting factors for eicosanoid production. In addition to the production of eicosanoids, PLA(2)-catalyzed membrane phospholipid hydrolysis is also the initiating step in the generation of PAF, a potent inflammatory agent. Thus, inhibition of PLA(2) activity should, in theory, be a more effective anti-inflammatory approach. However, developing an inhibitor that would be selective for the production of inflammatory metabolites and not inhibit the beneficial properties of PLA(2) has so far proved to be elusive. This review will focus on agents used currently to inhibit PLA(2) activity and will explore their possible therapeutic use.

Catalytic features, regulation and function of myocardial phospholipase A2.

Phospholipase A2 (PLA2) catalyzes the hydrolysis of sn-2 fatty acids from membrane phospholipids resulting in the production of several biologically active phospholipid metabolites such as lysophospholipids, arachidonic acid, eicosanoids and platelet-activating factor. The majority of myocardial PLA2 activity is membrane-associated and does not require Ca2+ for activity (iPLA2). Myocardial iPLA2 demonstrates unique characteristics when compared to other PLA2 isoforms described previously, including a selectivity for plasmalogen phospholipids and resistance to inhibition by methyl arachidonyl fluorophosphonate. Activation of myocardial iPLA2 results in the production of lysoplasmenylcholine and arachidonic acid, both of which can change the electrophysiologic properties of the myocardium. Arachidonic acid can modulate ion channel activity via protein kinase C activation and has been demonstrated to decrease gap junctional conductance. Lysoplasmenylcholine directly produces action potential derangements and alters calcium cycling in cardiac myocytes. Thus, inhibition of iPLA2 activity to block production of phospholipid metabolites that mediate pathologic changes in the myocardium would be of considerable benefit. However, there are situations where inhibition of PLA2 activity would be detrimental to the myocardium, in particular if iPLA2 acts as a phospholipid repair enzyme following oxidative damage. Although little is known regarding the function of cPLA2 or sPLA2 in the myocardium, it is possible that they may be important for signal transduction or may modulate the activity of iPLA2.

Endothelial cell PAF synthesis following thrombin stimulation utilizes Ca(2+)-independent phospholipase A(2).

Platelet activating factor (PAF) is a potent lipid autocoid that is rapidly synthesized and presented on the surface of endothelial cells following thrombin stimulation. PAF production may occur via de novo synthesis or by the combined direct action of phospholipase A(2) (PLA(2)) and acetyl-CoA:lyso-PAF acetyltransferase or via the remodeling pathway. This study was undertaken to define the role of PLA(2) and plasmalogen phospholipid hydrolysis in PAF synthesis in thrombin-treated human umbilical artery endothelial cells (HUAEC). Basal PLA(2) activity in HUAEC was primarily found to be Ca(2+)-independent (iPLA(2)), membrane-associated, and selective for arachidonylated plasmenylcholine substrate. Thrombin stimulation of HUAEC resulted in a preferential 3-fold increase in membrane-associated iPLA(2) activity utilizing plasmenylcholine substrates with a minimal increase in activity with alkylacyl glycerophospholipids. No change in cystolic iPLA(2) activity in thrombin-stimulated HUAEC was observed. The thrombin-stimulated activation of iPLA(2) and associated hydrolysis of plasmalogen phospholipids was accompanied by increased levels of arachidonic acid (from 1.1 +/- 0.1 to 2.8 +/- 0.1%) and prostacyclin release (from 38 +/- 12 to 512 +/- 24%) as well as an increased level of production of lysoplasmenylcholine (from 0.6 +/- 0.1 to 2.1 +/- 0.3 nmol/mg of protein), lysophosphatidylcholine (from 0.3 +/- 0.1 to 0.6 +/- 0.1 nmol/mg of protein), and PAF (from 790 +/- 108 to 3380 +/- 306 dpm). Inhibition of iPLA(2) with bromoenol lactone resulted in inhibition of iPLA(2) activity, plasmalogen phospholipid hydrolysis, production of choline lysophospholipids, and PAF synthesis. These data indicate that PAF production requires iPLA(2) activation in thrombin-stimulated HUAEC and may occur through the CoA-independent transacylase remodeling pathway rather than as a direct result of the PLA(2)-catalyzed hydrolysis of membrane alkylacyl glycerophosphocholine.

Clinical concentrations of doxorubicin inhibit activity of myocardial membrane-associated, calcium-independent phospholipase A(2).

Use of the anticancer antibiotic doxorubicin continues to be limited by its cumulative dose-related cardiotoxicity. Our study reports inhibition of myocardial intracellular calcium-independent phospholipase A(2) (iPLA(2)) activity by clinically relevant concentrations of the drug. The effect was first shown in vitro using suspensions of freshly isolated rat and rabbit cardiomyocytes. Addition of 0.1-10 microM doxorubicin to these cells led to a concentration- and time-dependent inhibition of total iPLA(2), as measured using (16:0, [(3)H]18:1) plasmenylcholine and phosphatidylcholine substrates in the presence or absence of calcium. Subcellular fractionation into cytosolic and membrane fraction revealed that the drug selectively inhibits membrane-associated iPLA(2) activity, without altering activity of the cytosolic enzyme. Doxorubicin treatment of cells prelabeled with [H(3)]arachidonic acid led to a depression of baseline arachidonic acid release levels, corroborating iPLA(2) inhibition. Reducing agents blocked PLA(2) inhibition in cardiomyocyte suspensions, suggesting that the doxorubicin effect is mediated by oxidation of susceptible cysteines. In vivo experiments, in which adults rats were i.v. injected with a bolus dose of 4 mg/kg doxorubicin, confirmed in vitro findings, revealing a 2-fold decrease in membrane-associated Ca(2+)-independent iPLA(2) activity in the heart tissue of treated animals. The observed phenomenon has important implications for myocyte signaling cascades and membrane remodeling.

Stimulation of protease activated receptors on RT4 cells mediates arachidonic acid release via Ca2+ independent phospholipase A2.

PURPOSE: Protease activated receptors (PAR) represent a family of G protein coupled receptors with 7 membrane spanning domains that are activated by proteolysis of the N-terminus of the receptor by serine proteases. The presence of multiple PARs on the same cell is thought to extend the range of proteases a cell responds to rather than expand the range of intracellular responses. We investigated arachidonic acid and prostaglandin E2 release in the human urothelial carcinoma cell line RT4 in response to stimulation with thrombin, which activates PAR-1, and tryptase, which activates PAR-2. MATERIALS AND METHODS: RT4 cells were incubated with thrombin, tryptase or PAR agonist peptides and intracellular phospholipase A2 (PLA2) activity, arachidonic acid and prostaglandin E2 release were measured. Pretreatment with bromoenol lactone, a selective inhibitor for Ca2+ independent PLA2 (iPLA2), was also investigated. RESULTS: Thrombin and tryptase stimulation resulted in a 2 to 3-fold increase in membrane associated iPLA2 that was accompanied by comparative increases in arachidonic acid and prostaglandin E2 release. These responses were also observed when synthetic peptides representing the tethered ligand for each receptor were incubated with RT4 cells. Arachidonic acid and prostaglandin E2 release, and iPLA2 activation were completely inhibited by pretreatment with bromoenol lactone. CONCLUSIONS: Stimulating RT4 cells with PAR-1 or PAR-2 leads to the selective activation of iPLA2 as well as the release of arachidonic acid and prostaglandin E2, which may provide cytoprotection during an acute inflammatory reaction.

Redistribution and abnormal activity of phospholipase A(2) isoenzymes in postinfarct congestive heart failure.

Cardiac sarcolemmal (SL) cis-unsaturated fatty acid sensitive phospholipase D (cis-UFA PLD) is modulated by SL Ca(2+)-independent phospholipase A(2) (iPLA(2)) activity via intramembrane release of cis-UFA. As PLD-derived phosphatidic acid influences intracellular Ca(2+) concentration and contractile performance of the cardiomyocyte, changes in iPLA(2) activity may contribute to abnormal function of the failing heart. We examined PLA(2) immunoprotein expression and activity in the SL and cytosol from noninfarcted left ventricular (LV) tissue of rats in an overt stage of congestive heart failure (CHF). Hemodynamic assessment of CHF animals showed an increase of the LV end-diastolic pressure with loss of contractile function. In normal hearts, immunoblot analysis revealed the presence of cytosolic PLA(2) (cPLA(2)) and secretory PLA(2) (sPLA(2)) in the cytosol, with cPLA(2) and iPLA(2) in the SL. Intracellular PLA(2) activity was predominantly Ca(2+) independent, with minimal sPLA(2) activity. CHF increased cPLA(2) immunoprotein and PLA(2) activity in the cytosol and decreased SL iPLA(2) and cPLA(2) immunoprotein and SL PLA(2) activity. sPLA(2) activity and abundance decreased in the cytosol and increased in SL in CHF. The results show that intrinsic to the pathophysiology of post-myocardial infarction CHF are abnormalities of SL PLA(2) isoenzymes, suggesting that PLA(2)-mediated bioprocesses are altered in CHF.

Oxidant-induced inhibition of myocardial calcium-independent phospholipase A2.

We discovered the acute inhibition of myocardial phospholipase A2 activity by micromolar concentrations of tert-butyl hydroperoxide and hydrogen peroxide. Specifically, freshly isolated adult rat cardiomyocytes were treated with the oxidants for 30 min, and phospholipase A2 activity was assessed in cell subcellular fractions using (16:0, [3H]18:1) plasmenylcholine and phosphatidylcholine substrates in the absence or presence of calcium. Calcium-independent phospholipase A2 activity was inhibited by approx 50% in both the cytosolic and membrane fractions by the oxidants. The inhibition of the phospholipase A2 activity was concentration dependent and preceded detectable changes in cell viability as assessed by lactate dehydrogenase release and rod-shaped morphology. Taking into account that oxidized sn-2 fatty acyl groups must first be hydrolyzed by phospholipase A2 to be repaired by glutathione peroxidase, we suggest that the observed inhibition of phospholipase A2 activity by oxidants compromises the myocyte's ability to deal with lipid peroxidation. This conclusion was further validated by the experiments in which pretreatment with the calcium-independent phospholipase A2 inhibitor bromoenol lactone exacerbated cardiotoxicity of tert-butyl hydroperoxide in myocyte cultures.

Phospholipase A(2)s in cell injury and death.

Phospholipase A(2)s (PLA(2)s) represent a family of esterases that hydrolyze the sn-2 ester bond in phospholipids, releasing free fatty acids and lysophospholipids. PLA(2)s are important in the signaling of several cellular processes and are known to play a significant role in inflammation. Studies also show that PLA(2)s are modulators of drug-, chemical-, and ischemia/reperfusion-induced cellular injury. The role of PLA(2)s in apoptosis and oncosis depends upon the PLA(2) isoform, the cell type, and the stimulus of injury. The purpose of this review is to discuss the functions of iPLA(2), cPLA(2) and sPLA(2) isoforms in oncosis and apoptosis, including oxidant-induced and receptor-mediated cell death. In addition, the measurement and modulation of PLA(2) is discussed.

Induction of Ca-independent PLA(2) and conservation of plasmalogen polyunsaturated fatty acids in diabetic heart.

Diabetes-induced changes in phospholipase A(2) (PLA(2)) activity have been measured in several tissues but are undefined in diabetic myocardium. We measured ventricular PLA(2) activity in control, streptozotocin-induced diabetic, and insulin-treated diabetic rats and characterized myocardial phospholipids to determine whether diabetes altered myocardial phospholipid metabolism. Increased membrane-associated Ca(2+)-independent PLA(2) (iPLA(2)) activity was observed in diabetes that was selective for arachidonylated phospholipids. Increased iPLA(2) activity was accompanied by an increase in choline lysophospholipids. Diabetes was associated with marked alterations in the phospholipid composition of the myocardium, characterized by decreases in esterified arachidonic and docosahexaenoic acids and increases in linoleic acid. The decrease in polyunsaturated fatty acids was confined to diacylphospholipids, whereas the relative amount of these fatty acids in plasmalogens was increased. Diabetes-induced changes in PLA(2) activity, lysophospholipid production, and alterations in phospholipid composition were all reversed by insulin treatment of diabetic animals. Diabetes-induced changes in membrane phospholipid content and phospholipid hydrolysis may contribute to some of the alterations in myocardial function that are observed in diabetic patients.

Selective plasmalogen substrate utilization by thrombin-stimulated Ca(2+)-independent PLA(2) in cardiomyocytes.

Thrombin stimulation of rabbit ventricular myocytes activates a membrane-associated, Ca(2+)-independent phospholipase A(2) (PLA(2)) capable of hydrolyzing plasmenylcholine (choline plasmalogen), plasmanylcholine (alkylacyl choline phospholipid), and phosphatidylcholine substrates. To identify the endogenous phospholipid substrates, we quantified the effects of thrombin stimulation on diradyl phospholipid mass and arachidonic acid and lysophospholipid production. Thrombin stimulation resulted in a selective decrease in arachidonylated plasmenylcholine, with no change in arachidonylated phosphatidylcholine. The decrease in arachidonylated plasmenylcholine was accompanied by an increase in plasmenylcholine species containing linoleic and linolenic acids at the sn-2 position. A decrease in arachidonylated plasmenylethanolamine was also observed after thrombin stimulation, with no concomitant change in arachidonylated phosphatidylethanolamine. Thrombin stimulation resulted in the selective production of lysoplasmenylcholine, with no increase in lysophosphatidylcholine content. There was no evidence for significant acetylation of lysophospholipids to form platelet-activating factor. Arachidonic acid released after thrombin stimulation was rapidly oxidized to prostacyclin. Thus thrombin-stimulated Ca(2+)-independent PLA(2) selectively hydrolyzes arachidonylated plasmalogen substrates, resulting in production of lysoplasmalogens and prostacyclin as the principal bioactive products.

Selective hydrolysis of plasmalogens in endothelial cells following thrombin stimulation.

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.

Stimulation of different phospholipase A2 isoforms by TNF-alpha and IL-1beta in adult rat ventricular myocytes.

We previously showed that in adult rat ventricular myocytes interleukin (IL)-1beta activates a membrane-associated, Ca2+-independent phospholipase A2 (iPLA2). In this study, we examined the possible existence of different PLA2 isoforms and effects of tumor necrosis factor (TNF)-alpha on iPLA2 activities. Western blot analysis identified iPLA2 in both membrane (approximately 82 kDa) and cytosolic (approximately 40 kDa) fractions and identified Ca2+-dependent PLA2 (cPLA2) only in cytosolic fractions. With plasmenylcholine or alkylacyl glycerophosphorylcholine as substrate, TNF-alpha elicited a twofold transient increase in cytosolic iPLA2 activity accompanied by an increase in arachidonic acid release and decreased membrane-associated iPLA2 activity with plasmenylcholine. With phosphatidylcholine as substrate, TNF-alpha decreased both cytosolic and membrane-associated iPLA2 activities. TNF-alpha-induced increases in cytosolic iPLA2 activity and arachidonic acid release were completely blocked by methyl arachidonyl fluorophosphonate (MAFP) but not by bromoenol lactone (BEL). TNF-alpha and IL-1beta together enhanced synergistically cytosolic and membrane PLA2 activities and arachidonic acid release that were blocked differentially by MAFP and BEL, respectively, and inhibited completely by MAFP plus BEL. These results suggest that TNF-alpha and IL-1beta act on different PLA2 isoforms in ventricular myocytes.

Selective hydrolysis of plasmalogen phospholipids by Ca2+-independent PLA2 in hypoxic ventricular myocytes.

Accelerated phospholipid catabolism occurs early after the onset of myocardial ischemia and is likely to be mediated by the activation of one or more phospholipases in ischemic tissue. We hypothesized that hypoxia increases phospholipase A2 (PLA2) activity in isolated ventricular myocytes, resulting in increased lysophospholipid and arachidonic acid production, contributing to arrhythmogenesis in ischemic heart disease. The majority of ventricular myocyte arachidonic acid was found in plasmalogen phospholipids. Hypoxia increased membrane-associated, Ca2+-independent, plasmalogen-selective PLA2 activity, resulting in increased arachidonic acid release and lysoplasmenylcholine production. Pretreatment with the specific Ca2+-independent PLA2 inhibitor bromoenol lactone blocked hypoxia-induced increases in PLA2 activity, arachidonic acid release, and lysoplasmenylcholine production. Lysoplasmenylcholine produced action potential derangements, including shortening of action potential duration, and induced early and delayed afterdepolarizations in normoxic myocytes. The electrophysiological alterations induced by lysoplasmenylcholine would likely contribute to the initiation of arrhythmogenesis in the ischemic heart.

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

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.

Calcium-independent phospholipase A2 in isolated rabbit ventricular myocytes.

We characterized phospholipase A2 (PLA2) activity in isolated rabbit ventricular myocytes with respect to subcellular distribution, substrate specificity, and Ca2+ dependency. Membrane-associated PLA2 was found to be an order of magnitude greater than cytosolic PLA2. Ventricular myocyte PLA2 activity was enhanced following protease-activated receptor stimulation with thrombin and was found to be largely Ca2+-independent and selective for phospholipid substrates containing arachidonic acid at the sn-2 position. Immunoblot analysis using an antibody to cytosolic Ca2+-independent PLA2 from Chinese hamster ovary cells recognized a membrane-associated protein with a molecular mass of approximately 80 kDa; however, differences in pH optima, response to inhibitors, and substrate selectivity of membrane-associated and cytosolic PLA2 activity suggest the presence of multiple Ca2+-independent PLA2. Pretreatment with bromoenol lactone, a specific inhibitor of Ca2+-independent PLA2, significantly attenuated membrane-associated and cytosolic PLA2 in unstimulated and thrombin-stimulated myocytes. Pretreatment with methyl arachidonyl fluorophosphonate, mepacrine, or dibucaine had no significant effect on PLA2 activity under all conditions tested. Ventricular myocyte PLA2 activity was significantly inhibited by ATP, GTP, and their nonhydrolyzable analogs and was regulated by protein kinase C activity. These studies demonstrate the presence of one or more unique membrane-associated Ca2+-independent PLA2 in isolated ventricular myocytes that exhibit a preference for phospholipids with arachidonate at the sn-2 position and that are activated by thrombin stimulation.

Lysophosphatidylcholine accumulation in cardiomyocytes requires thrombin activation of Ca2+-independent PLA2.

Lysophosphatidylcholine (LPC) accumulates during ischemia or following thrombin stimulation of cardiac myocytes. We determined whether LPC accumulation reflects increased LPC production via phospholipase A2 (PLA2) activation, inhibition of LPC catabolism, or a combination of both. Thrombin-stimulated normoxic myocytes demonstrated a 1.5-fold increase in LPC content and a 2- to 2.5-fold increase in membrane-associated, Ca2+-independent PLA2 activity. Despite PLA2 activation, hypoxia alone did not increase LPC content. Thrombin-stimulated hypoxic myocytes demonstrated a 2.5-fold increase in LPC content with no further increase in PLA2 activity. Inhibition of Ca2+-independent PLA2 prevented the thrombin-induced increase in both PLA2 activity and LPC content under normoxic and hypoxic conditions. Pharmacological blockade of the hypoxia-induced inhibition of LPC catabolism did not affect hypoxia or thrombin-induced PLA2 activation or normoxic, thrombin-induced LPC accumulation but greatly diminished the magnitude of LPC accumulation after thrombin stimulation of hypoxic myocytes. Thus accumulation of LPC during ischemia or after thrombin stimulation is absolutely dependent on PLA2 activation and further augmented by inhibition of LPC catabolism.