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.

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.

Neural dysfunction and metabolic imbalances in diabetic rats. Prevention by acetyl-L-carnitine.

The rationale for these experiments is that administration of L-carnitine and/or short-chain acylcarnitines attenuates myocardial dysfunction 1) in hearts from diabetic animals (in which L-carnitine levels are decreased); 2) induced by ischemia-reperfusion in hearts from nondiabetic animals; and 3) in nondiabetic humans with ischemic heart disease. The objective of these studies was to investigate whether imbalances in carnitine metabolism play a role in the pathogenesis of diabetic peripheral neuropathy. The major findings in rats with streptozotocin-induced diabetes of 4-6 weeks duration were that 24-h urinary carnitine excretion was increased approximately twofold and L-carnitine levels were decreased in plasma (46%) and sciatic nerve endoneurium (31%). These changes in carnitine levels/excretion were associated with decreased caudal nerve conduction velocity (10-15%) and sciatic nerve changes in Na(+)-K(+)-ATPase activity (decreased 50%), Mg(2+)-ATPase (decreased 65%), 1,2-diacyl-sn-glycerol (DAG) (decreased 40%), vascular albumin permeation (increased 60%), and blood flow (increased 65%). Treatment with acetyl-L-carnitine normalized plasma and endoneurial L-carnitine levels and prevented all of these metabolic and functional changes except the increased blood flow, which was unaffected, and the reduction in DAG, which decreased another 40%. In conclusion, these observations 1) demonstrate a link between imbalances in carnitine metabolism and several metabolic and functional abnormalities associated with diabetic polyneuropathy and 2) indicate that decreased sciatic nerve endoneurial ATPase activity (ouabain-sensitive and insensitive) in this model of diabetes is associated with decreased DAG.

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.

Subcellular distribution of endogenous long chain acylcarnitines during hypoxia in adult canine myocytes.

OBJECTIVE: Previous studies from our laboratory showed a pronounced increase in the sarcolemmal accumulation of long chain acylcarnitines in isolated neonatal rat myocytes after a prolonged (60 minute) hypoxic interval. Because much shorter intervals of hypoxia are associated with electrophysiological alterations in adult cells, the present study was performed to assess the extent of sarcolemmal accumulation of long chain acylcarnitines during hypoxia in adult canine myocytes. METHODS: Cells were incubated (for 24 hours) with 3H-carnitine and the uniformity of incorporation into carnitine fractions was verified biochemically. Cells were exposed to hypoxia (PO2 < 15 mm Hg) for 10 or 20 minutes in the presence or absence of sodium 2-[5-(4-chlorphenyl)-pentyl]-oxirane-2-carboxylate (POCA; 10 microM), an inhibitor of carnitine acyltransferase I. Cells were processed for electron microscopical autoradiography with a technique to spatially fix endogenous long chain acylcarnitines with selective and complete removal of short chain and free carnitine. Grain distributions were analysed by the maximum likelihood method from digitised micrographs. RESULTS: Total mass of long chain acylcarnitines increased ninefold [42.3(3.3) to 374(42) pmol.mg-1 protein] by 10 minutes of hypoxia and 15-fold [to 632(36) nmol.mg-1 protein] by 20 minutes of hypoxia. Normoxic cells exhibited little long chain acylcarnitines in the sarcolemma, and modest amounts in mitochondria and cytoplasm. In hypoxic cells, content of long chain acylcarnitines in mitochondria and cytoplasm increased by a maximum of twofold. By contrast, long chain acylcarnitines increased 100-fold in the sarcolemma to 4.18 x 10(6) molecules.microns-3 after 10 minutes of hypoxia. The increase in long chain acylcarnitines with hypoxia was completely prevented by pretreatment with POCA. CONCLUSION: Hypoxia in adult ventricular myocytes induces a rapid and preferential increase in endogenous long chain acylcarnitines within the sarcolemma.

Increased lysophosphatidylcholine content induced by thrombin receptor stimulation in adult rabbit cardiac ventricular myocytes.

OBJECTIVE: The aims were (1) to determine whether thrombin, which is increased in the presence of coronary thrombosis, can directly stimulate the production of lysophosphatidylcholine, which has arrhythmogenic properties, in ventricular myocytes; (2) whether the effect is dependent upon extracellular [Ca2+]; and (3) whether it is mediated directly through stimulation of the thrombin receptor. METHODS: Lipids were extracted from isolated adult rabbit ventricular myocytes and lysophosphatidylcholine was isolated by HPLC and quantified using a recently developed radiometric assay employing 3H-acetic anhydride. RESULTS: Thrombin (0.05 U.ml-1) stimulation of ventricular myocytes resulted in a nearly sixfold increase in lysophosphatidylcholine levels [0.26(SEM 0.03) to 1.61(0.42) nmol.mg-1 protein] within 1 min. The increase in myocytic lysophosphatidylcholine content was prevented by preincubation of thrombin with the proteolytic site inhibitors phenyly-prolyl-arginyl-chloromethyl ketone (PPACK) and dansylarginine N-(3-ethyl-1,5-pentanediyl)amide. The increase in lysophosphatidylcholine content in response to thrombin was not present at an extracellular calcium concentration ([Ca2+)]o) = 500 microM, but was marked at a physiological level of [Ca2+]o = 1.8 mM. Stimulation of myocytes with the thrombin receptor activating peptide SFLLRNPNDKYEPF (100 microM for 1 min) resulted in a similar increase in lysophosphatidylcholine content [1.61(0.27) nmol.mg-1 protein]. CONCLUSIONS: The marked increase in lysophosphatidylcholine content in cardiac myocytes in response to thrombin has important implications as an arrhythmogenic mechanism during early myocardial ischaemia.

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.

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.

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.

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.

Interleukin-1beta stimulates phospholipase A2 activity in adult rat ventricular myocytes.

We have examined whether interleukin (IL)-1beta modulates phospholipase A2 (PLA2) activity in ventricular myocytes. PLA2 activity was measured in isolated membrane and cytosol fractions with (16:0,[3H]18:1) plasmenylcholine and (16:0,[3H]18:1) phosphatidylcholine in the absence and presence of Ca2+. When measured in the absence of Ca2+ with plasmenylcholine, exposure to 5 ng/ml IL-1beta caused an increase in membrane-associated PLA2 activity for 10 min that returned to basal levels by 20 min. In the presence of Ca2+ with phosphatidylcholine, IL-1beta had no effect on membrane-associated PLA2 but decreased cytosolic PLA2 activity. Additionally, IL-1beta caused an increase in arachidonic acid release in 20 min. Pretreatment with E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one, a selective Ca2+-independent PLA2 inhibitor, blocked IL-1beta-induced increases in both PLA2 activity and arachidonic acid release. Exposure to IL-1 receptor antagonist (IL-1RA) alone had no effect on membrane-associated PLA2 activity. When incubated with IL-1beta, IL-1RA inhibited the IL-1beta-enhanced PLA2 activity. These results show that, via activation of its receptors, IL-1beta stimulates specifically membrane-associated Ca2+-independent plasmalogen-selective PLA2 in rat ventricular myocytes.

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.

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.

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.

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.

Thrombin-induced release of lysophosphatidylcholine from endothelial cells.

Lysophosphatidylcholine (LPC) increases extracellularly during ischemia in vivo in both animals and man as judged by measurements from venous effluents, but more recent studies have shown little or no increase in buffer-perfused, isolated heart preparations. The appearance of LPC in blood and lymph in animals and in venous effluents in man in response to ischemia suggests a vascular site for the production of LPC. The present study was performed to assess whether thrombin could stimulate phospholipase A2 in endothelial cells and whether this would evoke an increase in and release of LPC. Endothelial cells were disassociated from canine aortas by incubating with 0.1% collagenase for 20 min. Cells were plated and allowed to grow to confluence. Measurement of LPC was performed using Bligh and Dyer extraction of lipids, high performance liquid chromatography separation, and quantification of LPC using a recently developed radiometric assay employing [3H]acetic anhydride. Incubation of endothelial cells with thrombin (0.05 unit/ml) resulted in a 2.5-fold increase in LPC to 2.3 +/- 0.1 nmol/mg of protein at 2 min (p < 0.01) and returned to control levels within 20 min. The increase in LPC induced by thrombin exhibited a concentration-dependent response with an ED50 = 0.04 unit/ml. A concentration-dependent increase in LPC was also elicited by stimulation with the peptide portion of the thrombin receptor's tethered ligand SFLLRNPNDKYEPF with an ED50 = 8 microM. The LPC produced was rapidly and completely released into the surrounding media. Hirudin completely blocked the thrombin-induced increase in LPC. Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (0.1 microM), which rapidly inactivates thrombin's proteolytic activity in situ without impairing binding, or phenyl-prolyl-arginyl-chloromethyl ketone (PPACK, 5 nM), which inactivates thrombin due to chemical alteration of the proteolytic site, each prevented the increase in LPC in response to thrombin. Stimulation of protein kinase C with phorbol 12-myristate-13-acetate (PMA, 1 microM) enhanced the response to thrombin. In contrast, staurosporine (100 nM), H7 (15 microM), or chronic treatment with PMA for 20 h to down-regulate protein kinase C completely prevented the increase in LPC in response to thrombin. Thus, thrombin stimulation of endothelial cells in vivo during ischemia may be a primary mechanism contributing to the marked increase in LPC extracellularly during ischemia.

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.

Gradient elution reversed-phase chromatographic isolation of individual glycerophospholipid molecular species.

We describe a gradient elution reversed-phase high-performance liquid chromatographic approach for isolation of individual glycerophospholipid molecular species which greatly improves resolution and reduces run time compared to isocratic techniques. Separations were optimized and elution order and retention time data established by synthesizing 37 different homogeneous phospholipids comprising the major alkylacyl, diacyl and plasmalogen molecular species in samples derived from mammalian sources. Empirical equations which predict the elution order of individual species were derived. The method was validated with the use of complex mixtures of choline and ethanolamine glycerophospholipid species from isolated rabbit cardiomyocytes and porcine endothelial cells.