Calcium-independent phospholipase A2 is regulated by a novel protein kinase C in human coronary artery endothelial cells

We demonstrated previously that thrombin stimulation of endothelial cells activates a membrane-associated, Ca²⁺-independent phospholipase A₂ (iPLA₂) that selectively hydrolyzes arachidonylated plasmalogen phospholipids. We report that incubation of human coronary artery endothelial cells (HCAEC) with phorbol 12-myristate 13-acetate (PMA) to activate protein kinase C (PKC) resulted in hydrolysis of cellular phospholipids similar to that observed with thrombin stimulation (0.05 IU/ml; 10 min). Thrombin stimulation resulted in a decrease in arachidonylated plasmenylcholine (2.7 ± 0.1 vs. 5.3 ± 0.4 nmol PO₄/mg of protein) and plasmenylethanolamine (7.5 ± 1.0 vs. 12.0 ± 0.9 nmol PO₄/mg of protein). Incubation with PMA resulted in decreases in arachidonylated plasmenylcholine (3.2 ± 0.3 nmol PO₄/mg of protein) and plasmenylethanolamine (6.0 ± 1.0 nmol PO₄/mg of protein). Incubation of HCAEC with the selective iPLA₂ inhibitor bromoenol lactone (5 mM; 10 min) inhibited accelerated plasmalogen phospholipid hydrolysis in response to both PMA and thrombin stimulation. Incubation of HCAEC with PMA (100 nM; 5 min) resulted in increased arachidonic acid release (7.1 ± 0.3 vs. 1.1 ± 0.1%) and increased production of lysoplasmenylcholine (1.4 ± 0.2 vs. 0.6 ± 0.1 nmol PO₄/mg of protein), similar to the responses observed with thrombin stimulation. Downregulation of PKC by prolonged exposure to PMA (100 nM; 24 h) completely inhibited thrombin-stimulated increases in arachidonic acid release (7.1 ± 0.6 to 0.5 ± 0.1%) and lysoplasmenylcholine production (2.0 ± 0.1 to 0.2 ± 0.1 nmol PO₄/mg of protein). These data suggest that PKC activates iPLA₂ in HCAEC, leading to accelerated plasmalogen phospholipid hydrolysis and increased phospholipid metabolite production. lysophospholipids; cell signaling; phospholipid metabolism; arachidonic acid     

Calcium-independent phospholipase A2 mediates CREB phosphorylation in double-stranded RNA-stimulated endothelial cells

One of the products of a calcium-independent phospholipase A2 (iPLA2) attack of plasmenylcholine, lysoplasmenylcholine, has previously been shown to activate cAMP-dependent protein kinase (PKA). Because endothelial cells respond to some agonists in part by the activation of iPLA2, the present study was designed to determine whether double-stranded RNA (dsRNA), the primary activator of the antiviral response in endothelial cells, elicits cAMP response element binding protein (CREB) phosphorylation through a mechanism mediated by iPLA2. dsRNA stimulated CREB phosphorylation in bovine pulmonary artery endothelial cells that was inhibited by the iPLA2 inhibitor, bromoenol lactone, and the PKA inhibitor, H-89. Additionally, the product of iPLA2 hydrolysis of plasmenylcholine and lysoplasmenylcholine elicited CREB phosphorylation in bovine pulmonary endothelial cells. Taken together, the present studies suggest that dsRNA as well as other agonists of endothelial cells elicit signaling mechanisms that include in part CREB phosphorylation mediated by iPLA2.     

Calcium-independent phospholipase A2 mediates proliferation of human promonocytic U937 cells

We have investigated the possible involvement of two intracellular phospholipases A(2), namely group VIA calcium-independent phospholipase A(2) (iPLA(2)-VIA) and group IVA cytosolic phospholipase A(2) (cPLA(2)alpha), in the regulation of human promonocytic U937 cell proliferation. Inhibition of iPLA(2)-VIA activity by either pharmacological inhibitors such as bromoenol lactone or methyl arachidonyl fluorophosphonate or using specific antisense technology strongly blunted U937 cell proliferation. In contrast, inhibition of cPLA(2)alpha had no significant effect on U937 proliferation. Evaluation of iPLA(2)-VIA activity in cell cycle-synchronized cells revealed highest activity at G(2)/M and late S phases, and lowest at G(1). Phosphatidylcholine levels showed the opposite trend, peaking at G(1) and lowest at G(2)/M and late S phase. Reduction of U937 cell proliferation by inhibition of iPLA(2)-VIA activity was associated with arrest in G(2)/M and S phases. The iPLA(2)-VIA effects were found to be independent of the generation of free arachidonic acid or one of its oxygenated metabolites, and may work through regulation of the cellular level of phosphatidylcholine, a structural lipid that is required for cell growth/membrane expansion.     

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.     

Calcium-independent phospholipase A2 and apoptosis

Apoptosis or programmed cell death is associated with changes in glycerophospholipid metabolism. Cells undergoing apoptosis generally release free fatty acids including arachidonic acid, which parallels the reduction in cell viability. The involvement of cytosolic group IVA phospholipase A(2)alpha (cPLA(2)alpha) in apoptosis has been the subject of numerous studies but a clear picture of the role(s) played by this enzyme is yet to emerge. More recently, the importance of lipid products generated by the action of a second phospholipase A(2), the group VIA calcium-independent phospholipase A(2) (iPLA(2)-VIA) in apoptosis has begun to be unveiled. Current evidence suggests that iPLA(2)-VIA-derived lysophosphatidylcholine may play a prominent role in mediating the chemoattractant and recognition/engulfment signals that accompany the process of apoptotic cell death, and gives possibility to the efficient clearance of dying cells by circulating phagocytes. Other lines of evidence suggest that perturbations in the control of free arachidonic acid levels within the cells, a process that may implicate iPLA(2)-VIA as well, may provide important cellular signals for the onset of apoptosis.     

Calcium-independent phospholipase A2 in rat tissue cytosols

Cytosols (105,000 X g supernatant) from seven rat tissues were assayed for Ca2+-independent phospholipase A2 activity with either 1-acyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphocholine, 1-acyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphoethanolamine or 1-O-hexadecyl-2-[9,10-3H2]oleoyl-sn-glycero-3-phosphocholine as substrate. Low but consistent activities ranging from 10-120 pmol/min per mg protein were found in all tissues. The highest activities were present in liver, lung and brain. Total activities in mU/g wet weight were rather constant, ranging from 0.43 (heart) to 1.36 (liver). The soluble enzyme from rat lung cytosol was further investigated and was found to be capable of hydrolyzing microsomal membrane-associated substrates without exhibiting much selectivity for phosphatidylcholine species. Comparative gel filtration experiments of cytosol prepared from non-perfused and perfused lungs indicated that part of the Ca2+-independent phospholipase A2 originated from blood cells, but most of it was derived from lung cells. Lung cytosol also contained Ca2+-dependent phospholipase A2 activity, a small part of which originated from blood cells, presumably platelets. The major amount of Ca2+-dependent phospholipase A2 activity, however, came from lung cells. Neither this enzyme nor the Ca2+-independent phospholipase A2 from lung tissue showed immunological cross-reactivity with monoclonal antibodies against Ca2+-dependent phospholipase A2 isolated from rat liver mitochondria.     

Serum amyloid A induces endothelial dysfunction in porcine coronary arteries and human coronary artery endothelial cells

The objective of this study was to determine the effects and mechanisms of serum amyloid A (SAA) on coronary endothelial function. Porcine coronary arteries and human coronary arterial endothelial cells (HCAECs) were treated with SAA (0, 1, 10, or 25 microg/ml). Vasomotor reactivity was studied using a myograph tension system. SAA significantly reduced endothelium-dependent vasorelaxation of porcine coronary arteries in response to bradykinin in a concentration-dependent manner. SAA significantly decreased endothelial nitric oxide (NO) synthase (eNOS) mRNA and protein levels as well as NO bioavailability, whereas it increased ROS in both artery rings and HCAECs. In addition, the activities of internal antioxidant enzymes catalase and SOD were decreased in SAA-treated HCAECs. Bio-plex immunoassay analysis showed the activation of JNK, ERK2, and IkappaB-alpha after SAA treatment. Consequently, the antioxidants seleno-l-methionine and Mn(III) tetrakis-(4-benzoic acid)porphyrin and specific inhibitors for JNK and ERK1/2 effectively blocked the SAA-induced eNOS mRNA decrease and SAA-induced decrease in endothelium-dependent vasorelaxation in porcine coronary arteries. Thus, SAA at clinically relevant concentrations causes endothelial dysfunction in both porcine coronary arteries and HCAECs through molecular mechanisms involving eNOS downregulation, oxidative stress, and activation of JNK and ERK1/2 as well as NF-kappaB. These findings suggest that SAA may contribute to the progress of coronary artery disease.     

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

Accelerated phospholipid catabolism occurs early after the onsetof myocardial ischemia and is likely to be mediated by the activation of one or more phospholipases in ischemic tissue. We hypothesized that hypoxia increases phospholipaseA₂(PLA₂) activity in isolatedventricular myocytes, resulting in increased lysophospholipid andarachidonic acid production, contributing to arrhythmogenesis inischemic heart disease. The majority of ventricular myocyte arachidonicacid was found in plasmalogen phospholipids. Hypoxia increasedmembrane-associated,Ca²⁺-independent,plasmalogen-selective PLA₂activity, resulting in increased arachidonic acid release andlysoplasmenylcholine production. Pretreatment with the specificCa²⁺-independentPLA₂ inhibitor bromoenol lactoneblocked hypoxia-induced increases inPLA₂ activity, arachidonic acidrelease, and lysoplasmenylcholine production. Lysoplasmenylcholineproduced action potential derangements, including shortening of actionpotential duration, and induced early and delayed afterdepolarizationsin normoxic myocytes. The electrophysiological alterations induced bylysoplasmenylcholine would likely contribute to the initiation ofarrhythmogenesis in the ischemic heart.

     

Increasing plasmalogen levels protects human endothelial cells during hypoxia

Supplementation of cultured human pulmonary arterial endothelial cells (PAEC) with sn-1-O-hexadecylglycerol (HG) resulted in an approximately twofold increase in cellular levels of plasmalogens, a subclass of phospholipids known to have antioxidant properties; this was due, primarily, to a fourfold increase in the choline plasmalogens. Exposure of unsupplemented human PAEC to hypoxia (PO(2) = 20-25 mmHg) caused an increase in cellular reactive oxygen species (ROS) over a period of 5 days with a coincident decrease in viability. In contrast, HG-supplemented cells survived for at least 2 wk under these conditions with no evidence of increased ROS. Hypoxia resulted in a selective increase in the turnover of the plasmalogen plasmenylethanolamine. Human PAEC with elevated plasmalogen levels were also more resistant to H(2)O(2), hyperoxia, and the superoxide generator plumbagin. This protection was seemingly specific to cellular stresses in which significant ROS were generated because the sensitivity to lethal heat shock or glucose deprivation was not altered in HG-treated human PAEC. HG, by itself, was not sufficient for protection; HG supplementation of bovine PAEC had no effect upon plasmalogen levels and did not rescue these cells from the cytotoxic effects of hypoxia. This is the initial demonstration that plasmalogen content can be substantially enhanced in a normal cell. These data also demonstrate that HG can protect cells during hypoxia and other ROS-mediated stress, likely due to the resulting increase in these antioxidant phospholipids.     

Bombesin stimulates the rapid activation of phospholipase A2-catalyzed phosphatidylcholine hydrolysis in Swiss 3T3 cells.

In Swiss 3T3 fibroblasts bombesin stimulated the release of arachidonic acid in a time- and dose-dependent manner. Arachidonate levels were significantly elevated after only a 2-s stimulation with the agonist. Furthermore, by measuring the arachidonate content of cellular phospholipids after cell activation, it was shown that there was selective depletion from phosphatidylcholine over the same time course. The corresponding production of lysophosphatidylcholine suggested the involvement of a phosphatidylcholine-specific phospholipase A2. Initial arachidonic acid release was not dependent on the presence of extracellular calcium, not activated by treatment of the cells with thapsigargin, and was unaffected by down-regulation of protein kinase C activity, or by treatment of the cells with the protein kinase C inhibitor staurosporine. These data strongly suggest that occupation of the bombesin receptor is closely coupled to activation of phospholipase A2 which results in the rapid release of arachidonic acid from phosphatidylcholine.     

Epinephrine upregulates superoxide dismutase in human coronary artery endothelial cells

Regular exercise resulting in release of catecholamines is an oxidant stress, and yet it protects humans from acute cardiac events. We designed this study to examine the effect of epinephrine on free radical release and endogenous superoxide dismutase (SOD) gene and protein expression in human coronary artery endothelial cells (HCAECs). HCAECs were incubated with epinephrine (10(-9) to 10(-5) M) alone or with the water-soluble analog of vitamin E (trolox) (10(-5) M), the lipid-soluble vitamin E (5 x 10(-5) M), or the beta(1)-adrenergic blocker atenolol (10(-5) M). At 1 and 24 h of incubation with epinephrine, superoxide anion generation increased by 102 and 81% in the HCAECs. There was a marked increase in both MnSOD and Cu/ZnSOD mRNA and protein, as determined by RT-PCR and Western Analysis, respectively. Both MnSOD and Cu/ZnSOD activities were also increased. Pretreatment of HCAECs with trolox and vitamin E decreased superoxide anion generation (p <.05 vs. epinephrine alone) and blocked the subsequent upregulation of SOD mRNA and protein. Treatment of cells with the beta-blocker atenolol also blocked the upregulation of SOD (p <.05 vs. epinephrine alone). These observations suggest that epinephrine via beta(1)-adrenoceptor activation causes superoxide anion generation, and the superoxide subsequently upregulates the endogenous antioxidant species SOD. These observations may be the basis of long-term benefits of exercise.     

Calcium-independent phospholipase A(2): structure and function

The classical Ca(2+)-independent phospholipase A(2) enzyme, now known as Group VIA PLA(2), was initially purified and characterized from the P388D(1) macrophage-like cell line. The corresponding cDNA was subsequently cloned from a variety of sources, and it is now known that multiple splice variants of the enzyme are expressed, some of which may act as negative regulators of the active enzyme. Group VIA PLA(2) has a consensus lipase motif (GTSTG) containing the catalytic serine, is 85-88 kDa, and exists in an aggregated form. The enzyme contains multiple ankyrin repeats, which may play a role in oligomerization. The Group VIA enzyme exhibits lysophospholipase activity as well as phospholipase A(2) activity, and it is capable of hydrolyzing a wide variety of phospholipid substrates. A major function of Group VIA PLA(2) is to mediate phospholipid remodeling, but the enzyme may play other roles as well. Other Ca(2+)-independent PLA(2) enzymes have more recently been identified, and it may be possible to discriminate between the various Ca(2+)-independent PLA(2) enzymes based on sequence or inhibitor-sensitivity. However, the physiological functions of the newly identified enzymes have yet to be elucidated.     

Fas induces apoptosis in human coronary artery endothelial cells in vitro

Background  

Published work suggests that some types of endothelial cells undergo apoptosis in response to ligation of the receptor Fas (CD95, APO1) but other types are resistant. Because heterogeneity among endothelial cells from different tissues, has been demonstrated, the purpose of this study was to determine, if Fas ligation and/or activation by human Fas ligand induces apoptosis and caspase activities, in cultured human coronary artery endothelial cells, and the differences between TNF-a and FAS induced apoptosis in these cells.     

Lysoplasmenylcholine increases neutrophil adherence to human coronary artery endothelial cells

We demonstrated previously that thrombin stimulation of human coronary artery endothelial cells (HCAEC) results in release of choline lysophospholipids [lysophosphatidylcholine (lysoPtdCho) and lysoplasmenylcholine (lysoPlsCho)]. These amphiphilic metabolites have been implicated in arrhythmogenesis following the onset of myocardial ischemia, but studies examining their direct effects on the vasculature remain limited. We and others have shown that thrombin and lysoPtdCho can increase cell surface adhesion molecules and adherence of circulating inflammatory cells to the endothelium. This study supports our hypothesis that these changes may be mediated, at least in part, by lysoPlsCho, thus implicating this metabolite as an inflammatory mediator in the coronary vasculature and a modulator of the progression of atherosclerosis. Apical stimulation of HCAEC with thrombin resulted in the production and release of choline lysophospholipids from the apical surface of the HCAEC monolayer. Basolateral stimulation had no effect on choline lysophospholipid production or release from either the apical or basolateral surface of the HCAEC monolayer. Incubation of HCAEC with lysoPlsCho or lysoPtdCho resulted in similar increases in HCAEC surface expression of P-selectin and E-selectin. Furthermore, lysoPlsCho increased cell surface expression of P-selectin, E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 with a time course similar to that of thrombin stimulation. Increased presence of cell surface adhesion molecules may contribute to the significant increase in adherence of neutrophils to either thrombin- or lysoPlsCho-stimulated HCAEC. These results demonstrate that the presence of thrombin at sites of vascular injury in the coronary circulation, resulting in increased choline lysophospholipid release from the HCAEC apical surface, has the potential to propagate vascular inflammation by upregulation of adhesion molecules and recruitment of circulating inflammatory cells to the endothelium. endothelium; arrhythmogenesis; inflammation; lysophospholipids     

Calcium and magnesium dependence of phospholipase A2-catalyzed hydrolysis of phosphatidylcholine small unilamellar vesicles.

The Ca2+ requirement for lipid hydrolysis catalyzed by phospholipase A2 from Agkistrodon piscivorus piscivorus (App-D49) and porcine pancreas has been examined using small, unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC SUV). Hydrolysis was affected by product inhibition even at early times, and the extent of this inhibition depended on the concentration of divalent cations. The Ca2+ requirement for half-maximal rates of hydrolysis reflected, in part, this non-catalytic role of divalent cations. The presence of 10 mM Mg2+, a cation which does not support catalysis, reduced the Ca2+ required for half-maximal rates of hydrolysis from millimolar concentrations to 40 microM for App-D49. Since the dissociation constant of the enzyme for Ca2+ in solution is 2 mM, these results indicate a change in the interaction of the enzyme with Ca2+ under catalytic conditions. The kinetic dissociation constant of Ca2+ for the pancreatic enzyme was 20 microM which is substantially lower than the dissociation constant in solution, 0.35 mM. The similarity of apparent kinetic dissociation constants for these enzymes suggests that structurally similar features determine the affinity for Ca2+ under catalytic conditions. Evidence is presented that the affinity of phospholipase A2 for Ca2+ changes subsequent to the initial interaction of the enzyme with the substrate interface. However, the apparent Michaelis constant, KMapp, for App-D49, 0.03-0.06 mM, is independent of [Ca2+] and is about the same as the equilibrium dissociation constant for DPPC SUV, 0.14 mM. We thus suggest that KMapp is a steady-state constant.     

Calcium-independent phospholipase A2 modulates cytosolic oxidant activity and contractile function in murine skeletal muscle cells.

Phospholipase A2 (PLA2) activity supports production of reactive oxygen species (ROS) by mammalian cells. In skeletal muscle, endogenous ROS modulate the force of muscle contraction. We tested the hypothesis that skeletal muscle cells constitutively express the calcium-independent PLA2 (iPLA2) isoform and that iPLA2 modulates both cytosolic oxidant activity and contractile function. Experiments utilized differentiated C2C12 myotubes and a panel of striated muscles isolated from adult mice. Muscle preparations were processed for measurement of mRNA by real-time PCR, protein by immunoblot, cytosolic oxidant activity by the dichlorofluorescein oxidation assay, and contractile function by in vitro testing. We found that iPLA2 was constitutively expressed by all muscles tested (myotubes, diaphragm, soleus, extensor digitorum longus, gastrocnemius, heart) and that mRNA and protein levels were generally similar among muscles. Selective iPLA2 blockade by use of bromoenol lactone (10 microM) decreased cytosolic oxidant activity in myotubes and intact soleus muscle fibers. iPLA2 blockade also inhibited contractile function of unfatigued soleus muscles, shifting the force-frequency relationship rightward and depressing force production during acute fatigue. Each of these changes could be reproduced by selective depletion of superoxide anions using superoxide dismutase (1 kU/ml). These findings suggest that constitutively expressed iPLA2 modulates oxidant activity in skeletal muscle fibers by supporting ROS production, thereby influencing contractile properties and fatigue characteristics.