Roles of phospholipase A2 isoforms in swelling- and melittin-induced arachidonic acid release and taurine efflux in NIH3T3 fibroblasts

Osmotic swelling of NIH3T3 mouse fibroblasts activates a bromoenol lactone (BEL)-sensitive taurine efflux, pointing to the involvement of a Ca²⁺-independent phospholipase A₂ (iPLA₂) (Lambert IH. J Membr Biol 192: 19–32, 2003). We report that taurine efflux from NIH3T3 cells was not only increased by cell swelling but also decreased by cell shrinkage. Arachidonic acid release to the cell exterior was similarly decreased by shrinkage yet not detectably increased by swelling. NIH3T3 cells were found to express cytosolic calcium-dependent cPLA₂-IVA, cPLA₂-IVB, cPLA₂-IVC, iPLA₂-VIA, iPLA₂-VIB, and secretory sPLA₂-V. Arachidonic acid release from swollen cells was partially inhibited by BEL and by the sPLA₂-inhibitor manoalide. Cell swelling elicited BEL-sensitive arachidonic acid release from the nucleus, to which iPLA₂-VIA localized. Exposure to the bee venom peptide melittin, to increase PLA₂ substrate availability, potentiated arachidonic acid release and osmolyte efflux in a volume-sensitive, 5-lipoxygenase-dependent, cyclooxygenase-independent manner. Melittin-induced arachidonic acid release was inhibited by manoalide and slightly but significantly by BEL. A BEL-sensitive, melittin-induced PLA₂ activity was also detected in lysates devoid of sPLA₂, indicating that both sPLA₂ and iPLA₂ contribute to arachidonic acid release in vivo. Swelling-induced taurine efflux was inhibited potently by BEL and partially by manoalide, whereas the reverse was true for melittin-induced taurine efflux. It is suggested that in NIH3T3 cells, swelling-induced taurine efflux is dependent at least in part on arachidonic acid release by iPLA₂ and possibly also by sPLA₂, whereas melittin-induced taurine efflux is dependent on arachidonic acid release by sPLA₂ and, to a lesser extent, iPLA₂. osmotic stress; cell volume regulation; calcium-independent phospholipase A₂; secretory phospholipase A₂; nucleus     

Specific differential expression of phospholipase A2 subtypes in rat cerebellum

Phospholipase A₂ (PLA₂) is a family of enzymes playing diverse roles in lipid signaling in neurons and glia cells. In this study, we examined the expression of subtypes of PLA₂ in the cerebellum using immunolabeling and in situ hybridization methods. Two Ca²⁺-dependent cytosolic subtypes (cPLA₂α and cPLA₂β), one Ca²⁺-independent cytosolic subtype (iPLA₂), and two secretory subtypes (sPLA₂IIA and sPLA₂V) were detected in the cerebellum. cPLA₂α is present in somata and dendrites of Purkinje cells, while sPLA₂IIA is associated with the endoplasmic reticulum in perinuclear regions of Purkinje cell somata. iPLA₂ is present in granule cells, stellate cells and also in the nucleus of Purkinje cells. In addition, cPLA₂β is localized in granule cells, and sPLA₂V in Bergmann glia cells. These results provide an important basis for identifying functional roles of PLA₂s in the cerebellum.     

Ca2+ sensitivity of BK channels in GH3 cells involves cytosolic phospholipase A2

To test thehypothesis that intracellular Ca²⁺activation of large-conductanceCa²⁺-activatedK⁺ (BK) channels involves thecytosolic form of phospholipase A₂ (cPLA₂), we first inhibited theexpression of cPLA₂ by treating GH₃ cells with antisenseoligonucleotides directed at the two possible translation start siteson cPLA₂. Western blot analysis and a biochemical assay of cPLA₂activity showed marked inhibition of the expression ofcPLA₂ in antisense-treated cells.We then examined the effects of intracellularCa²⁺ concentration([Ca²⁺]_i)on single BK channels from these cells. Open channel probability (P_o) for thecells exposed to cPLA₂ antisenseoligonucleotides in 0.1 µM intracellularCa²⁺ was significantly lower thanin untreated or sense oligonucleotide-treated cells, but the voltagesensitivity did not change (measured as the slope of theP_o-voltagerelationship). In fact, a 1,000-fold increase in[Ca²⁺]_ifrom 0.1 to 100 µM did not significantly increaseP_oin these cells, whereas BK channels from cells in the other treatmentgroups showed a normalP_o-[Ca²⁺]_iresponse. Finally, we examined the effect of exogenous arachidonic acidon theP_oof BK channels from antisense-treated cells. Although arachidonic aciddid significantly increaseP_o,it did so without restoring the[Ca²⁺]_isensitivity observed in untreated cells. We conclude that although [Ca²⁺]_idoes impart some basal activity to BK channels inGH₃ cells, the steepP_o-[Ca²⁺]_irelationship that is characteristic of these channels involves cPLA₂.

     

Recent progress in phospholipase A₂ research: from cells to animals to humans

Mammalian genomes encode genes for more than 30 phospholipase A₂s (PLA₂s) or related enzymes, which are subdivided into several classes including low-molecular-weight secreted PLA₂s (sPLA₂s), Ca²⁺-dependent cytosolic PLA₂s (cPLA₂s), Ca²⁺-independent PLA₂s (iPLA₂s), platelet-activating factor acetylhydrolases (PAF-AHs), lysosomal PLA₂s, and a recently identified adipose-specific PLA. Of these, the intracellular cPLA₂ and iPLA₂ families and the extracellular sPLA₂ family are recognized as the “big three”. From a general viewpoint, cPLA₂α (the prototypic cPLA₂) plays a major role in the initiation of arachidonic acid metabolism, the iPLA₂ family contributes to membrane homeostasis and energy metabolism, and the sPLA₂ family affects various biological events by modulating the extracellular phospholipid milieus. The cPLA₂ family evolved along with eicosanoid receptors when vertebrates first appeared, whereas the diverse branching of the iPLA₂ and sPLA₂ families during earlier eukaryote development suggests that they play fundamental roles in life-related processes. During the past decade, data concerning the unexplored roles of various PLA₂ enzymes in pathophysiology have emerged on the basis of studies using knockout and transgenic mice, the use of specific inhibitors, and information obtained from analysis of human diseases caused by mutations in PLA₂ genes. This review focuses on current understanding of the emerging biological functions of PLA₂s and related enzymes.     

Assessing Phospholipase A2 Activity toward Cardiolipin by Mass Spectrometry

Cardiolipin, a major component of mitochondria, is critical for mitochondrial functioning including the regulation of cytochrome c release during apoptosis and proper electron transport. Mitochondrial cardiolipin with its unique bulky amphipathic structure is a potential substrate for phospholipase A₂ (PLA₂) in vivo. We have developed mass spectrometric methodology for analyzing PLA₂ activity toward various cardiolipin forms and demonstrate that cardiolipin is a substrate for sPLA₂, cPLA₂ and iPLA₂, but not for Lp-PLA₂. Our results also show that none of these PLA₂s have significant PLA₁ activities toward dilyso-cardiolipin. To understand the mechanism of cardiolipin hydrolysis by PLA₂, we also quantified the release of monolyso-cardiolipin and dilyso-cardiolipin in the PLA₂ assays. The sPLA₂s caused an accumulation of dilyso-cardiolipin, in contrast to iPLA₂ which caused an accumulation of monolyso-cardiolipin. Moreover, cardiolipin inhibits iPLA₂ and cPLA₂, and activates sPLA₂ at low mol fractions in mixed micelles of Triton X-100 with the substrate 1-palmitoyl-2-arachidonyl-sn-phosphtidylcholine. Thus, cardiolipin functions as both a substrate and a regulator of PLA₂ activity and the ability to assay the various forms of PLA₂ is important in understanding its function.     

Expression of phospholipases A2 in primary human lung macrophages: Role of cytosolic phospholipase A2-α in arachidonic acid release and platelet activating factor synthesis

Macrophages are a major source of lipid mediators in the human lung. Expression and contribution of cytosolic (cPLA₂) and secreted phospholipases A₂ (sPLA₂) to the generation of lipid mediators in human macrophages are unclear. We investigated the expression and role of different PLA₂s in the production of lipid mediators in primary human lung macrophages. Macrophages express the alpha, but not the zeta isoform of group IV and group VIA cPLA₂ (iPLA₂). Two structurally-divergent inhibitors of group IV cPLA₂ completely block arachidonic acid release by macrophages in response to non-physiological (Ca²⁺ ionophores and phorbol esters) and physiological agonists (lipopolysaccharide and Mycobacterium protein derivative). These inhibitors also reduce by 70% the synthesis of platelet-activating factor by activated macrophages. Among the full set of human sPLA₂s, macrophages express group IIA, IID, IIE, IIF, V, X and XIIA, but not group IB and III enzymes. Me-Indoxam, a potent and cell impermeable inhibitor of several sPLA₂s, has no effect on arachidonate release or platelet-activating factor production. Agonist-induced exocytosis is not influenced by cPLA₂ inhibitors at concentrations that block arachidonic acid release. Our results indicate that human macrophages express cPLA₂-alpha, iPLA₂ and several sPLA₂s. Cytosolic PLA₂-alpha is the major enzyme responsible for lipid mediator production in human macrophages.     

Activation of cytosolic phospholipase A2 in permeabilized human neutrophils

Neutrophils (PMN) contain two types of phospholipase A₂ (PLA₂), a 14 kDa ‘secretory’ Type II PLA₂ (sPLA₂) and an 85 kDa ‘cytosolic’ PLA₂ (cPLA₂), that differ in a number of key characteristics: (1) cPLA₂ prefers arachidonate (AA) as a substrate but hydrolyzes all phospholipids; sPLA₂ is not AA specific but prefers ethanolamine containing phosphoacylglycerols. (2) cPLA₂ is active at nM calcium (Ca²⁺) concentrations; sPLA₂ requires μM Ca²⁺ levels. (3) cPLA₂ activity is regulated by phosphorylation; sPLA₂ lacks phosphorylation sites. (4) cPLA₂ is insensitive to reduction; sPLA₂ is inactivated by agents that reduce disulfide bonds. We utilized PMN permeabilized with Staphylococcus aureus α-toxin to determine whether one or both forms of PLA₂ were activated in porated cells under conditions designed to differentiate between the two enzymes. PMN were labeled with [³H]AA to measure release from phosphatidylcholine and phosphatidylinositol; gas chromatography-mass spectrometry was utilized to determine total AA release (mainly from phosphatidylethanolamine) and to asses oleate and linoleate mass. A combination of 500 nM Ca²⁺, a guanine nucleotide, and stimulation with n-formyl-met-leu-phe (FMLP) were necessary to induce maximal AA release in permeabilized PMN measured by either method; AA was preferentially released. [³H]AA and AA mass release occurred in parallel over time. A hydrolyzable form of ATP was necessary for maximum AA release and staurosporin inhibited PLA₂ activation. Dithiothreitol treatment had little affect on [³H]AA release and metabolism but inhibited AA mass release. Assay of cell supernatants after cofactor addition did not detect sPLA₂ activity and the cytosolic buffer utilized did not support activity of recombinant sPLA₂. These results strongly suggested that cPLA₂ was the enzyme activated in the permeabilized cell model and this is the first report which unambiguously demonstrates AA release in response to activation of a specific type of PLA₂ in PMN.     

Activation of MAPKs in thrombin-stimulated ventricular myocytes is dependent on Ca2+-independent PLA2

Thrombin stimulation of isolated rabbit ventricular myocytes activates a membrane-associated, Ca²⁺-independent PLA₂ (iPLA₂) that selectively hydrolyzes plasmalogen phospholipids and results in increased production of arachidonic acid and lysoplasmenylcholine. To determine whether MAPK regulates myocardial iPLA₂ activity, we isolated ventricular myocytes from rabbit heart by collagenase digestion and pretreated them with MAPK inhibitors before stimulating them with thrombin. Pretreatment with PD-98059 to inhibit p42/44 MAPK or SB-203580 to inhibit p38 MAPK had no significant effect on thrombin-stimulated, membrane-associated iPLA₂ activity. Thrombin stimulation resulted in significant increases in both p42/44 and p38 MAPK activity after 2 min. Pretreatment with the iPLA₂-selective inhibitor bromoenol lactone completely inhibited thrombin-stimulated MAPK activity, suggesting that activation of MAPKs was dependent on iPLA₂ activation. Ventricular myocyte MAPK activity was increased by incubation of the myocytes with lysoplasmenylcholine, a metabolite produced by iPLA₂-catalyzed membrane plasmalogen phospholipid hydrolysis. Altogether, these data suggest that activation of MAPKs occurs downstream of and is dependent on iPLA₂ activation in thrombin-stimulated rabbit ventricular myocytes. lysoplasmenylcholine; cell signaling; protease-activated receptors     

Activities and interactions among phospholipases A2 during thapsigargin-induced S49 cell death

The purpose of this study was to determine the roles of calcium-dependent phospholipase A₂ (cPLA₂) and calcium-independent phospholipase A₂ (iPLA₂) in thapsigargin-induced membrane susceptibility to secretory phospholipase A₂ (sPLA₂) and programmed cell death. ³H-arachidonic acid release was observed in the presence of thapsigargin. This release was inhibited partially by an inhibitor of iPLA₂ (BEL) and completely by an inhibitor of both cPLA₂ and iPLA₂ (MAFP) suggesting that these enzymes were active during apoptosis. The process of cell death did not require the activity of either enzyme since neither inhibitor impeded the progression of apoptosis. However, both inhibitors increased the susceptibility of the membrane to sPLA₂ in the presence of thapsigargin. In the case of BEL, this effect appeared to involve direct induction of apoptosis in a sub-population of the cells independent of the action of iPLA₂. In conclusion, the results suggested that cPLA₂ and iPLA₂ are active during thapsigargin-induced apoptosis in S49 cells and that cPLA₂ tempers the tendency of the cells to become susceptible to sPLA₂ during apoptosis.     

Calcium dependence of C-type natriuretic peptide-formed fast K+ channel

The lipid bilayertechnique was used to characterize theCa²⁺ dependence of a fastK⁺ channel formed by a synthetic17-amino acid segment [OaCNP-39-(1-17)] ofa 39-amino acid C-type natriuretic peptide (OaCNP-39) found in platypus (Ornithorhynchusanatinus) venom (OaV). TheOaCNP-39-(1-17)-formed K⁺ channel was reversiblydependent on1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-buffered cis (cytoplasmic)Ca²⁺ concentration([Ca²⁺]_cis).The channel was fully active when[Ca²⁺]_ciswas >10⁴ M andtrans (luminal)Ca²⁺ concentration was 1.0 mM, butnot at low[Ca²⁺]_cis.The open probability of single channels increased from zero at1 × 10⁶ McisCa²⁺ to 0.73 ± 0.17 (n = 22) at10³ McisCa²⁺. Channel openings to themaximum conductance of 38 pS were rapidly and reversibly activated when[Ca²⁺]_cis,but not transCa²⁺ concentration(n = 5), was increased to >5 × 10⁴ M(n = 14). Channel openings to thesubmaximal conductance of 10.5 pS were dominant at5 × 10⁴ MCa²⁺.K⁺ channels did not open whencisMg²⁺ orSr²⁺ concentrations were increasedfrom zero to 10³ M or when[Ca²⁺]_ciswas maintained at 10⁶ M(n = 3 and 2). The Hill coefficientand the inhibition constant were 1 and 0.8 × 10⁴ McisCa²⁺, respectively. Thisdependence of the channel on high[Ca²⁺]_cissuggests that it may become active under1) physiological conditions whereCa²⁺ levels are high, e.g., duringcardiac and skeletal muscle contractions, and2) pathological conditions that leadto a Ca²⁺ overload, e.g., ischemicheart and muscle fatigue. The channel could modify a cascade ofphysiological functions that are dependent on theCa²⁺-activatedK⁺ channels, e.g., vasodilationand salt secretion.

     

Selective hydrolysis of plasmalogens in endothelial cells following thrombin stimulation

The presentstudy was performed to characterize thrombin-stimulated phospholipaseA₂(PLA₂) activity and theresultant release of lysophospholipids from endothelial cells. Themajority of PLA₂ activity inendothelial cells was membrane associated,Ca²⁺ independent, and arachidonateselective. Incubation with thrombin increased membrane-associatedPLA₂ activity using bothplasmenylcholine and alkylacyl glycerophosphocholine substrates in theabsence of Ca²⁺, with no increasein activity observed with phosphatidylcholine substrate. The increasedPLA₂ activity was accompanied byarachidonic acid and lysoplasmenylcholine (LPlasC) release fromendothelial cells into the surrounding medium. Thrombin-induced changeswere duplicated by stimulation with the thrombin-receptor-directed peptide SFLLRNPNDKYEPF. Pretreatment with theCa²⁺-independentPLA₂ inhibitor bromoenol lactoneblocked thrombin-stimulated increases inPLA₂ activity, arachidonic acid,and LPlasC release. Stimulation of protein kinase C (PKC) increasedbasal PLA₂ activity and LPlasCproduction. Thrombin-stimulatedPLA₂ activity and LPlasC production were enhanced with PKC activation and completely prevented with PKC downregulation. Thus thrombin treatment of endothelial cellsactivates a PKC-activated, membrane-associated,Ca²⁺-independentPLA₂ that selectively hydrolyzesarachidonylated, ether-linked phospholipid substrates, resulting inLPlasC and arachidonic acid release.

     

Activation and inactivation of the volume-sensitive taurine leak pathway in NIH3T3 fibroblasts and Ehrlich Lettre ascites cells

Hypotonic exposure provokes the mobilization of arachidonic acid, production of ROS, and a transient increase in taurine release in Ehrlich Lettre cells. The taurine release is potentiated by H₂O₂ and the tyrosine phosphatase inhibitor vanadate and reduced by the phospholipase A₂ (PLA₂) inhibitors bromoenol lactone (BEL) and manoalide, the 5-lipoxygenase (5-LO) inhibitor ETH-615139, the NADPH oxidase inhibitor diphenyl iodonium (DPI), and antioxidants. Thus, swelling-induced taurine efflux in Ehrlich Lettre cells involves Ca²⁺-independent (iPLA₂)/secretory PLA₂ (sPLA₂) plus 5-LO activity and modulation by ROS. Vanadate and H₂O₂ stimulate arachidonic acid mobilization and vanadate potentiates ROS production in Ehrlich Lettre cells and NIH3T3 fibroblasts under hypotonic conditions. However, vanadate-induced potentiation of the volume-sensitive taurine efflux is, in both cell types, impaired in the presence of BEL and DPI and following restoration of the cell volume. Thus, potentiation of the volume-sensitive taurine efflux pathway following inhibition of tyrosine phosphatase activity reflects increased arachidonic acid mobilization and ROS production for downstream signaling. Vanadate delays the inactivation of volume-sensitive taurine efflux in NIH3T3 cells, and this delay is impaired in the presence of DPI. Vanadate has no effect on the inactivation of swelling-induced taurine efflux in Ehrlich Lettre cells. It is suggested that increased tyrosine phosphorylation of regulatory components of NADPH oxidase leads to increased ROS production and a subsequent delay in inactivation of the volume-sensitive taurine efflux pathway and that NADPH oxidase or antioxidative capacity differ between NIH3T3 and Ehrlich Lettre cells. organic osmolytes; reactive oxygen species; vanadate; H₂O₂; tyrosine phosphatases; arachidonic acid mobilization     

Macrophage arachidonate release via both the cytosolic Ca2+-dependent and -independent phospholipases is necessary for cell spreading

We have observed that phospholipase A₂ (PLA₂) activation and arachidonate (AA) release are essential for monocyte/macrophage adherence and spreading. In this study, we addressed the relationship between AA release and cell adherence/spreading in murine resident peritoneal macrophages, and the roles of specific PLA₂s in these processes. The PLA₂-specific inhibitors, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL, specific for the Ca²⁺-independent PLA₂ (iPLA₂)) and methyl arachidonoyl fluorophosphonate (MAFP, specific for the Ca²⁺-dependent phospholipase (cPLA₂)) inhibited AA release and cell spreading in a correlated fashion but only modestly decreased cell adherence. Cell spreading was normalized by the addition of AA to PLA₂-inhibited cells. AA release during spreading was also inhibited by Ca²⁺ depletion or protein kinase C (PKC) inhibition, and was accompanied by increased (but transient) phosphorylation of cPLA₂. Inhibition of macrophage spreading, however, only partially inhibited AA release. Moreover, constitutive AA release was seen in fully spread macrophages which was inhibited by BEL, but not MAFP or Ca²⁺ depletion. BEL also reversed the phenotype of fully spread cells. These data suggest that macrophage spreading requires the release of AA by the iPLA₂ (which appears to be constitutively active) and cPLA₂ (which appears to be stimulated by adherence/spreading). Maintenance of macrophage spreading, in contrast, appears to be principally dependent on the iPLA₂.     

Cu2+-induced modification of the kinetics of A beta(1-42) channels

We found that the amyloid peptide A(1-42) is capable of interacting with membrane and forming heterogeneous ion channels in the absence of any added Cu²⁺ or biological redox agents that have been reported to mediate A(1-42) toxicity. The A(1-42)-formed cation channel was inhibited by Cu²⁺ in cis solution ([Cu²⁺]_cis) in a voltage- and concentration-dependent manner between 0 and 250 µM. The [Cu²⁺]_cis-induced channel inhibition is fully reversible at low concentrations between 50 and 100 µM [Cu²⁺]_cis and partially reversible at 250 µM [Cu²⁺]_cis. The inhibitory effects of [Cu²⁺]_cis between 50 and 250 µM on the channel could not be reversed with addition of Cu²⁺-chelating agent clioquinol (CQ) at concentrations between 64 and 384 µM applied to the cis chamber. The effects of 200-250 µM [Cu²⁺]_cis on the burst and intraburst kinetic parameters were not fully reversible with either wash or 128 µM [CQ]_cis. The kinetic analysis of the data indicate that Cu²⁺-induced inhibition was mediated via both desensitization and an open channel block mechanism and that Cu²⁺ binds to the histidine residues located at the mouth of the channel. It is proposed that the Cu²⁺-binding site of the A(1-42)-formed channels is modulated with Cu²⁺ in a similar way to those of channels formed with the prion protein fragment PrP(106-126), suggesting a possible common mechanism for Cu²⁺ modulation of A and PrP channel proteins linked to neurodegenerative diseases. neurodegenerative diseases; transitional metals; ion channel pathologies; membrane injuries; calcium homeostasis     

Phospholipase A2 catalysis and lipid mediator lipidomics

Phospholipase A₂ (PLA₂) enzymes are the upstream regulators of the eicosanoid pathway liberating free arachidonic acid from the sn-2 position of membrane phospholipids. Free intracellular arachidonic acid serves as a substrate for the eicosanoid biosynthetic enzymes including cyclooxygenases, lipoxygenases, and cytochrome P450s that lead to inflammation. The Group IVA cytosolic (cPLA₂), Group VIA calcium-independent (iPLA₂), and Group V secreted (sPLA₂) are three well-characterized human enzymes that have been implicated in eicosanoid formation. In this review, we will introduce and summarize the regulation of catalytic activity and cellular localization, structural characteristics, interfacial activation and kinetics, substrate specificity, inhibitor binding and interactions, and the downstream implications for eicosanoid biosynthesis of these three important PLA₂ enzymes.     

New potent and selective polyfluoroalkyl ketone inhibitors of GVIA calcium-independent phospholipase A2

Group VIA calcium-independent phospholipase A₂ (GVIA iPLA₂) has recently emerged as an important pharmaceutical target. Selective and potent GVIA iPLA₂ inhibitors can be used to study its role in various neurological disorders. In the current work, we explore the significance of the introduction of a substituent in previously reported potent GVIA iPLA₂ inhibitors. 1,1,1,2,2-Pentafluoro-7-(4-methoxyphenyl)heptan-3-one (GK187) is the most potent and selective GVIA iPLA₂ inhibitor ever reported with a X_I(50) value of 0.0001, and with no significant inhibition against GIVA cPLA₂ or GV sPLA₂. We also compare the inhibition of two difluoromethyl ketones on GVIA iPLA₂, GIVA cPLA₂, and GV sPLA₂.     

ANG II-induced translocation of cytosolic PLA2 to the nucleus in vascular smooth muscle cells

The accumulation of radiolabeled arachidonicacid (AA), immunoblot analysis of subcellular fractions, andimmunofluorescence tagging of proteins in intact cells were used toexamine the coupling of ANG II receptors with the activity and locationof a cytosolic phospholipase A₂(cPLA₂) in vascular smoothmuscle cells (VSMC). ANG II induced the accumulation of AA, whichpeaked by 10 min and was downregulated by 20 min. A large proportion ofthe AA released in response to ANG II was due to the activation of a Ca²⁺-dependent lipase coupled toan AT₁ receptor. However,regulation of Ca²⁺ availabilityfailed to completely block AA release, and a small but significantreduction in ANG II-mediated AA release was observed in the presence ofan AT₂ antagonist. These findings,coupled with a 25% reduction in the ANG II-induced AA release by aninhibitor specific for aCa²⁺-independentPLA₂, are consistent with thepresence and activation of aCa²⁺-independentPLA₂. In contrast, immunoblotanalysis and immunofluorescence detection showed that the ANGII-mediated translocation of cPLA₂ to a membrane fraction was exclusivelyAT₁ dependent and regulated byCa²⁺ availability. Furthermore,the nucleus was the membrane target. We conclude that ANG II regulatesthe Ca²⁺-dependent activation andtranslocation of cPLA₂ through anAT₁ receptor and that this eventis targeted at the nucleus in VSMC.

     

Regulation of membrane-associated iPLA2 activity by a novel PKC isoform in ventricular myocytes

Thrombin stimulation of rabbit ventricularmyocytes increases membrane-associated, Ca²⁺-independentphospholipase A₂ (iPLA₂) activity, resulting inaccelerated hydrolysis of membrane plasmalogen phospholipids andincreased production of arachidonic acid and lysoplasmenylcholine. This study was designed to investigate the signal transduction pathways involved in activation of membrane-associated iPLA₂.Incubation of isolated membrane fractions suspended inCa²⁺-free buffer with thrombin or phorbol 12-myristate13-acetate resulted in a two- to threefold increase iniPLA₂ activity. Prior treatment with the PKC inhibitorGF-109203X blocked iPLA₂ activation by thrombin. These datasuggest that a novel PKC isoform present in the membrane fractionmodulates iPLA₂ activity. Immunoblot analysis revealed asignificant portion of PKC- present in the membrane fraction, but noother membrane-associated novel PKC isoform was detected by thismethod. These data indicate that activation of membrane-associatediPLA₂ is mediated by a membrane-associated novel PKCisoform in thrombin-stimulated rabbit ventricular myocytes.

     

Complex regulation of store-operated Ca2+ entry pathway by PKC-{varepsilon} in vascular SMCs

The role of PKC in the regulation of store-operated Ca²⁺ entry (SOCE) is rather controversial. Here, we used Ca²⁺-imaging, biochemical, pharmacological, and molecular techniques to test if Ca²⁺-independent PLA₂β (iPLA₂β), one of the transducers of the signal from depleted stores to plasma membrane channels, may be a target for the complex regulation of SOCE by PKC and diacylglycerol (DAG) in rabbit aortic smooth muscle cells (SMCs). We found that the inhibition of PKC with chelerythrine resulted in significant inhibition of thapsigargin (TG)-induced SOCE in proliferating SMCs. Activation of PKC by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused a significant depletion of intracellular Ca²⁺ stores and triggered Ca²⁺ influx that was similar to TG-induced SOCE. OAG and TG both produced a PKC-dependent activation of iPLA₂β and Ca²⁺ entry that were absent in SMCs in which iPLA₂β was inhibited by a specific chiral enantiomer of bromoenol lactone (S-BEL). Moreover, we found that PKC regulates TG- and OAG-induced Ca²⁺ entry only in proliferating SMCs, which correlates with the expression of the specific PKC- isoform. Molecular downregulation of PKC- impaired TG- and OAG-induced Ca²⁺ influx in proliferating SMCs but had no effect in confluent SMCs. Our results demonstrate that DAG (or OAG) can affect SOCE via multiple mechanisms, which may involve the depletion of Ca²⁺ stores as well as direct PKC--dependent activation of iPLA₂β, resulting in a complex regulation of SOCE in proliferating and confluent SMCs. protein kinase C-; Ca²⁺-independent phospholipase A₂; diacylglycerol; smooth muscle cells