Calcium-independent Phospholipase A2β Research Articles

Lysophosphatidylserine induces necrosis in pressure overloaded male mouse hearts via G protein coupled receptor 34

Heart failure is a leading cause of mortality in developed countries. Cell death is a key player in the development of heart failure. Calcium-independent phospholipase A2β (iPLA2β) produces lipid mediators by catalyzing lipids and induces nuclear shrinkage in caspase-independent cell death. Here, we show that lysophosphatidylserine generated by iPLA2β induces necrotic cardiomyocyte death, as well as contractile dysfunction mediated through its receptor, G protein-coupled receptor 34 (GPR34). Cardiomyocyte-specific iPLA2β-deficient male mice were subjected to pressure overload. While control mice showed left ventricular systolic dysfunction with necrotic cardiomyocyte death, iPLA2β-deficient mice preserved cardiac function. Lipidomic analysis revealed a reduction of 18:0 lysophosphatidylserine in iPLA2β-deficient hearts. Knockdown of Gpr34 attenuated 18:0 lysophosphatidylserine-induced necrosis in neonatal male rat cardiomyocytes, while the ablation of Gpr34 in male mice reduced the development of pressure overload-induced cardiac remodeling. Thus, the iPLA2β—lysophosphatidylserine—GPR34—necrosis signaling axis plays a detrimental role in the heart in response to pressure overload.

Lipidome-wide 13C flux analysis: a novel tool to estimate the turnover of lipids in organisms and cultures

Lipid metabolism plays an important role in the regulation of cellular homeostasis. However, because it is difficult to measure the actual rates of synthesis and degradation of individual lipid species, lipid compositions are often used as a surrogate to evaluate lipid metabolism even though they provide only static snapshots of the lipodome. Here, we designed a simple method to determine the turnover rate of phospholipid and acylglycerol species based on the incorporation of 13C6-glucose combined with LC-MS/MS. We labeled adult Drosophila melanogaster with 13C6-glucose that incorporates into the entire lipidome, derived kinetic parameters from mass spectra, and studied effects of deletion of CG6718, the fly homolog of the calcium-independent phospholipase A2β, on lipid metabolism. Although 13C6-glucose gave rise to a complex pattern of 13C incorporation, we were able to identify discrete isotopomers in which 13C atoms were confined to the glycerol group. With these isotopomers, we calculated turnover rate constants, half-life times, and fluxes of the glycerol backbone of multiple lipid species. To perform these calculations, we estimated the fraction of labeled molecules in glycerol-3-phosphate, the lipid precursor, by mass isotopomer distribution analysis of the spectra of phosphatidylglycerol. When we applied this method to D. melanogaster, we found a range of lipid half-lives from 2 to 200 days, demonstrated tissue-specific fluxes of individual lipid species, and identified a novel function of CG6718 in triacylglycerol metabolism. This method provides fluxomics-type data with significant potential to improve the understanding of complex lipid regulation in a variety of research models.

Cellular Plasmalogen Content Does Not Influence Arachidonic Acid Levels or Distribution in Macrophages: A Role for Cytosolic Phospholipase A2γ in Phospholipid Remodeling.

Availability of free arachidonic acid (AA) constitutes a rate limiting factor for cellular eicosanoid synthesis. AA distributes differentially across membrane phospholipids, which is largely due to the action of coenzyme A-independent transacylase (CoA-IT), an enzyme that moves the fatty acid primarily from diacyl phospholipid species to ether-containing species, particularly the ethanolamine plasmalogens. In this work, we examined the dependence of AA remodeling on plasmalogen content using the murine macrophage cell line RAW264.7 and its plasmalogen-deficient variants RAW.12 and RAW.108. All three strains remodeled AA between phospholipids with similar magnitude and kinetics, thus demonstrating that cellular plasmalogen content does not influence the process. Cell stimulation with yeast-derived zymosan also had no effect on AA remodeling, but incubating the cells in AA-rich media markedly slowed down the process. Further, knockdown of cytosolic-group IVC phospholipase A2γ (cPLA2γ) by RNA silencing significantly reduced AA remodeling, while inhibition of other major phospholipase A2 forms such as cytosolic phospholipase A2α, calcium-independent phospholipase A2β, or secreted phospholipase A2 had no effect. These results uncover new regulatory features of CoA-IT-mediated transacylation reactions in cellular AA homeostasis and suggest a hitherto unrecognized role for cPLA2γ in maintaining membrane phospholipid composition via regulation of AA remodeling.

SLPI Inhibits ATP-Mediated Maturation of IL-1β in Human Monocytic Leukocytes: A Novel Function of an Old Player

Interleukin-1β (IL-1β) is a potent, pro-inflammatory cytokine of the innate immune system that plays an essential role in host defense against infection. However, elevated circulating levels of IL-1β can cause life-threatening systemic inflammation. Hence, mechanisms controlling IL-1β maturation and release are of outstanding clinical interest. Secretory leukocyte protease inhibitor (SLPI), in addition to its well-described anti-protease function, controls the expression of several pro-inflammatory cytokines on the transcriptional level. In the present study, we tested the potential involvement of SLPI in the control of ATP-induced, inflammasome-dependent IL-1β maturation and release. We demonstrated that SLPI dose-dependently inhibits the ATP-mediated inflammasome activation and IL-1β release in human monocytic cells, without affecting the induction of pro-IL-1β mRNA by LPS. In contrast, the ATP-independent IL-1β release induced by the pore forming bacterial toxin nigericin is not impaired, and SLPI does not directly modulate the ion channel function of the human P2X7 receptor heterologously expressed in Xenopus laevis oocytes. In human monocytic U937 cells, however, SLPI efficiently inhibits ATP-induced ion-currents. Using specific inhibitors and siRNA, we demonstrate that SLPI activates the calcium-independent phospholipase A2β (iPLA2β) and leads to the release of a low molecular mass factor that mediates the inhibition of IL-1β release. Signaling involves nicotinic acetylcholine receptor subunits α7, α9, α10, and Src kinase activation and results in an inhibition of ATP-induced caspase-1 activation. In conclusion, we propose a novel anti-inflammatory mechanism induced by SLPI, which inhibits the ATP-dependent maturation and secretion of IL-1β. This novel signaling pathway might lead to development of therapies that are urgently needed for the prevention and treatment of systemic inflammation.

Alpha-1 Antitrypsin Inhibits ATP-Mediated Release of Interleukin-1β via CD36 and Nicotinic Acetylcholine Receptors.

While interleukin (IL)-1β is a potent pro-inflammatory cytokine involved in host defense, high levels can cause life-threatening sterile inflammation including systemic inflammatory response syndrome. Hence, the control of IL-1β secretion is of outstanding biomedical importance. In response to a first inflammatory stimulus such as lipopolysaccharide, pro-IL-1β is synthesized as a cytoplasmic inactive pro-form. Extracellular ATP originating from injured cells is a prototypical second signal for inflammasome-dependent maturation and release of IL-1β. The human anti-protease alpha-1 antitrypsin (AAT) and IL-1β regulate each other via mechanisms that are only partially understood. Here, we demonstrate that physiological concentrations of AAT efficiently inhibit ATP-induced release of IL-1β from primary human blood mononuclear cells, monocytic U937 cells, and rat lung tissue, whereas ATP-independent IL-1β release is not impaired. Both, native and oxidized AAT are active, suggesting that the inhibition of IL-1β release is independent of the anti-elastase activity of AAT. Signaling of AAT in monocytic cells involves the lipid scavenger receptor CD36, calcium-independent phospholipase A2β, and the release of a small soluble mediator. This mediator leads to the activation of nicotinic acetylcholine receptors, which efficiently inhibit ATP-induced P2X7 receptor activation and inflammasome assembly. We suggest that AAT controls ATP-induced IL-1β release from human mononuclear blood cells by a novel triple-membrane-passing signaling pathway. This pathway may have clinical implications for the prevention of sterile pulmonary and systemic inflammation.

The structure of iPLA2\u03b2 reveals dimeric active sites and suggests mechanisms of regulation and localization

Calcium-independent phospholipase A2β (iPLA2β) regulates important physiological processes including inflammation, calcium homeostasis and apoptosis. It is genetically linked to neurodegenerative disorders including Parkinson’s disease. Despite its known enzymatic activity, the mechanisms underlying iPLA2β-induced pathologic phenotypes remain poorly understood. Here, we present a crystal structure of iPLA2β that significantly revises existing mechanistic models. The catalytic domains form a tight dimer. They are surrounded by ankyrin repeat domains that adopt an outwardly flared orientation, poised to interact with membrane proteins. The closely integrated active sites are positioned for cooperative activation and internal transacylation. The structure and additional solution studies suggest that both catalytic domains can be bound and allosterically inhibited by a single calmodulin. These features suggest mechanisms of iPLA2β cellular localization and activity regulation, providing a basis for inhibitor development. Furthermore, the structure provides a framework to investigate the role of neurodegenerative mutations and the function of iPLA2β in the brain.

Progressive Axonal Degeneration of Nigrostriatal Dopaminergic Neurons in Calcium-Independent Phospholipase A2β Knockout Mice.

Calcium-independent phospholipase A2β (iPLA2β, PLA2G6) is essential for the remodeling of membrane glycerophospholipids. Mutations in this gene are responsible for autosomal recessive, young onset, L-dopa-responsive parkinsonism (PARK14), suggesting a neurodegenerative condition in the nigrostriatal dopaminergic system in patients with PLA2G6 mutations. We previously observed slowly progressive motor deficits in iPLA2β-knockout (KO) mice. To clarify whether a deficiency of iPLA2β leads to the degeneration of nigrostriatal dopaminergic neurons, we analyzed the striatum of iPLA2β-KO mice. At all clinical stages, nerve terminals in the striatum were immunopositive for tyrosine hydroxylase (TH) and dopamine transporter (DAT) in wild-type (WT) control mice. In iPLA2β-KO mice, focal loss of nerve terminals positive for TH and DAT was found from 56 weeks (early clinical stage), although iPLA2β-KO mice at 56 weeks showed no significant decrease in the number of dopaminergic neurons in the substantia nigra compared with age-matched WT mice, as reported previously. At 100 weeks (late clinical stage), greater decreases in DAT immunoreactivity were observed in the striatum of iPLA2β-KO mice. Moreover, strongly TH-positive structures, presumed to be deformed axons, were observed in the neuropils of the striatum of iPLA2β-KO mice starting at 15 weeks (preclinical stage) and increased with age. These results suggest that the degeneration of dopaminergic neurons occurs mainly in the distal region of axons in iPLA2β-KO mice.

Progesterone-induced Acrosome Exocytosis Requires Sequential Involvement of Calcium-independent Phospholipase A2β (iPLA2β) and Group X Secreted Phospholipase A2 (sPLA2)

Phospholipase A2 (PLA2) activity has been shown to be involved in the sperm acrosome reaction (AR), but the molecular identity of PLA2 isoforms has remained elusive. Here, we have tested the role of two intracellular (iPLA2β and cytosolic PLA2α) and one secreted (group X) PLA2s in spontaneous and progesterone (P4)-induced AR by using a set of specific inhibitors and knock-out mice. iPLA2β is critical for spontaneous AR, whereas both iPLA2β and group X secreted PLA2 are involved in P4-induced AR. Cytosolic PLA2α is dispensable in both types of AR. P4-induced AR spreads over 30 min in the mouse, and kinetic analyses suggest the presence of different sperm subpopulations, using distinct PLA2 pathways to achieve AR. At low P4 concentration (2 μm), sperm undergoing early AR (0-5 min post-P4) rely on iPLA2β, whereas sperm undergoing late AR (20-30 min post-P4) rely on group X secreted PLA2. Moreover, the role of PLA2s in AR depends on P4 concentration, with the PLA2s being key actors at low physiological P4 concentrations (≤2 μm) but not at higher P4 concentrations (~10 μm).

Increased susceptibility to bladder inflammation in smokers: targeting the PAF-PAF receptor interaction to manage inflammatory cell recruitment.

Chronic bladder inflammation can result in a significant reduction in quality of life. Smoking remains a leading preventable risk factor in many diseases. Despite the large amount of evidence supporting the risks of smoking, roughly 45 million people in the United States remain smokers. The impact of cigarette smoking on inflammation is well established, but how smoking promotes bladder inflammation is currently unknown. The aim of this study was to determine if cigarette smoke exposure impacts inflammatory cell adherence to bladder endothelial cells and if targeting the platelet-activating factor (PAF)-PAF receptor (PAFR) interaction could be beneficial in managing bladder inflammation. In response to cigarette smoke extract (CSE) incubation, bladder endothelial cells from human or mouse displayed increased PAF accumulation, decreased PAF-AH activity, and increased inflammatory cell adherence. Inhibition of endothelial cell calcium-independent phospholipase A2β (iPLA2β) with (S)-BEL, to block PAF production, prevented adherence of inflammatory cells. Pretreatment of inflammatory cells with PAFR antagonists, ginkgolide B or WEB2086 significantly reduced the number of adhered cells to bladder endothelium. Wild-type mice exposed to cigarette smoke displayed increased presence of inflammatory infiltration which was absent in iPLA2β(-/-) mice and those exposed to room air. In conclusion, cigarette smoke exposure increases endothelial cell PAF accumulation and increased inflammatory cell adherence. Inhibition of PAF accumulation or PAFR antagonism markedly attenuated inflammatory cell adherence to bladder endothelial cells. The results detailed in this study highlight to potential therapeutic targets for managing bladder inflammation.

Enhanced breast cancer cell adherence to the lung endothelium via PAF acetylhydrolase inhibition: a potential mechanism for enhanced metastasis in smokers.

Cancer deaths are primarily caused by distant metastases, rather than by primary tumor growth; however, the role of smoking in metastasis remains unclear. We demonstrated previously that endothelial cell platelet-activating factor (PAF) production results in enhanced inflammatory cell recruitment to the lung. We propose that endothelial cell PAF accumulation plays a role in cancer cell migration to distal locations. We used cigarette smoke extract (CSE) to inhibit the activity of endothelial cell PAF acetylhydrolase (PAF-AH), which hydrolyzes and inactivates PAF, and determined whether this results in increased endothelial cell PAF accumulation and breast cancer adherence. Incubation of human lung microvascular endothelial cells (HMVEC-L) with CSE resulted in a significant inhibition of PAF-AH activity that was accompanied by increased PAF production and adherence of highly invasive MDA-MB-231 breast cancer cells. Pretreatment of HMVEC-L with (S)-bromoenol lactone to inhibit calcium-independent phospholipase A2β (iPLA2β, which initiates endothelial cell PAF production) prior to CSE exposure resulted in complete inhibition of MDA-MB-231 cell adherence. Similarly, pretreatment of MDA-MB-231 cells with the PAF receptor antagonist Ginkgo biloba resulted in inhibition of adherence to the endothelium. Immunoblot analysis indicated an increase in MDA-MB-231 cell PAF receptor expression with CSE exposure. Taken together, our data indicate that CSE exposure increases endothelial cell PAF production, resulting in enhanced adherence of tumor cells to the endothelium. Our in vitro data indicate that increased tumor cell adherence would lead to enhanced metastasis formation in smokers. Potential therapeutic targets include endothelial cell iPLA2β or the tumor cell PAF receptor.

Mechanism-Based Inhibition of iPLA2β Demonstrates a Highly Reactive Cysteine Residue (C651) That Interacts with the Active Site: Mass Spectrometric Elucidation of the Mechanisms Underlying Inhibition

The multifaceted roles of calcium-independent phospholipase A2β (iPLA2β) in numerous cellular processes have been extensively examined through utilization of the iPLA2-selective inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL). Herein, we employed accurate mass/high resolution mass spectrometry to demonstrate that the active site serine (S465) and C651 of iPLA2β are covalently cross-linked during incubations with BEL demonstrating their close spatial proximity. This cross-link results in macroscopic alterations in enzyme molecular geometry evidenced by anomalous migration of the cross-linked enzyme by SDS-PAGE. Molecular models of iPLA2β constructed from the crystal structure of iPLA2α (patatin) indicate that the distance between S465 and C651 is approximately 10 Å within the active site of iPLA2β. Kinetic analysis of the formation of the 75 kDa iPLA2β-BEL species with the (R) and (S) enantiomers of BEL demonstrated that the reaction of (S)-BEL with iPLA2β was more rapid than for (R)-BEL paralleling the enantioselectivity for the inhibition of catalysis by each inhibitor with iPLA2β. Moreover, we demonstrate that the previously identified selective acylation of iPLA2β by oleoyl-CoA occurs at C651 thereby indicating the importance of active site architecture for acylation of this enzyme. Collectively, these results identify C651 as a highly reactive nucleophilic residue within the active site of iPLA2β which is thioesterified by BEL, acylated by oleoyl-CoA, and located in close spatial proximity to the catalytic serine thereby providing important chemical insights on the mechanisms through which BEL inhibits iPLA2β and the topology of the active site.

Smooth Muscle-specific Expression of Calcium-independent Phospholipase A2β (iPLA2β) Participates in the Initiation and Early Progression of Vascular Inflammation and Neointima Formation

Whether group VIA phospholipase A(2) (iPLA(2)β) is involved in vascular inflammation and neointima formation is largely unknown. Here, we report that iPLA(2)β expression increases in the vascular tunica media upon carotid artery ligation and that neointima formation is suppressed by genetic deletion of iPLA(2)β or by inhibiting its activity or expression via perivascular delivery of bromoenol lactone or of antisense oligonucleotides, respectively. To investigate whether smooth muscle-specific iPLA(2)β is involved in neointima formation, we generated transgenic mice in which iPLA(2)β is expressed specifically in smooth muscle cells and demonstrate that smooth muscle-specific expression of iPLA(2)β exacerbates ligation-induced neointima formation and enhanced both production of proinflammatory cytokines and vascular infiltration by macrophages. With cultured vascular smooth muscle cell, angiotensin II, arachidonic acid, and TNF-α markedly induce increased expression of IL-6 and TNF-α mRNAs, all of which were suppressed by inhibiting iPLA(2)β activity or expression with bromoenol lactone, antisense oligonucleotides, and genetic deletion, respectively. Similar suppression also results from genetic deletion of 12/15-lipoxygenase or inhibiting its activity with nordihydroguaiaretic acid or luteolin. Expression of iPLA(2)β protein in cultured vascular smooth muscle cells was found to depend on the phenotypic state and to rise upon incubation with TNF-α. Our studies thus illustrate that smooth muscle cell-specific iPLA(2)β participates in the initiation and early progression of vascular inflammation and neointima formation and suggest that iPLA(2)β may represent a novel therapeutic target for preventing cardiovascular diseases.

P4.14 Investigating the role of calcium-independent phospholipase [formula omitted] in store-operated calcium entry in mdx muscle

Duchenne muscular dystrophy (DMD) is caused by lack of dystrophin. Characteristic signs of dystrophic muscles are membrane fragility and abnormally elevated levels of intracellular calcium, detrimental to the cells. Increased activity of store-operated calcium channels (SOC) has been proposed as a possible mechanism for the enhanced calcium concentration. Studies of SOC have indicated the enzyme calcium-independent phospholipase A2β (iPLA2β) as an important mediator of calcium influx. We have previously shown that SOC entry in FDB fibres isolated from mdx mice is reduced in the presence of bromoenol lactone (BEL), a suicide inhibitor of iPLA2.

Neuroaxonal Dystrophy in Calcium-Independent Phospholipase A2β Deficiency Results from Insufficient Remodeling and Degeneration of Mitochondrial and Presynaptic Membranes

Infantile neuroaxonal dystrophy (INAD) is a fatal neurodegenerative disease characterized by the widespread presence of axonal swellings (spheroids) in the CNS and PNS and is caused by gene abnormality in PLA2G6 [calcium-independent phospholipase A(2)β (iPLA(2)β)], which is essential for remodeling of membrane phospholipids. To clarify the pathomechanism of INAD, we pathologically analyzed the spinal cords and sciatic nerves of iPLA(2)β knock-out (KO) mice, a model of INAD. At 15 weeks (preclinical stage), periodic acid-Schiff (PAS)-positive granules were frequently observed in proximal axons and the perinuclear space of large neurons, and these were strongly positive for a marker of the mitochondrial outer membrane and negative for a marker of the inner membrane. By 100 weeks (late clinical stage), PAS-positive granules and spheroids had increased significantly in the distal parts of axons, and ultrastructural examination revealed that these granules were, in fact, mitochondria with degenerative inner membranes. Collapse of mitochondria in axons was accompanied by focal disappearance of the cytoskeleton. Partial membrane loss at axon terminals was also evident, accompanied by degenerative membranes in the same areas. Imaging mass spectrometry showed a prominent increase of docosahexaenoic acid-containing phosphatidylcholine in the gray matter, suggesting insufficient membrane remodeling in the presence of iPLA(2)β deficiency. Prominent axonal degeneration in neuroaxonal dystrophy might be explained by the collapse of abnormal mitochondria after axonal transportation. Insufficient remodeling and degeneration of mitochondrial inner membranes and presynaptic membranes appear to be the cause of the neuroaxonal dystrophy in iPLA(2)β-KO mice.

Platelet-activating factor and metastasis: calcium-independent phospholipase A2β deficiency protects against breast cancer metastasis to the lung

We determined the contribution of calcium-independent phospholipase A(2)β (iPLA(2)β) to lung metastasis development following breast cancer injection into wild-type (WT) and iPLA(2)β-knockout (iPLA(2)β-KO) mice. WT and iPLA(2)β-KO mice were injected in the mammary pad with 200,000 E0771 breast cancer cells. There was no difference in primary tumor size between WT and iPLA(2)β-KO mice at 27 days postinjection. However, we observed an 11-fold greater number of breast cancer cells in the lungs of WT mice compared with iPLA(2)β-KO animals (P < 0.05). Isolated WT lung endothelial cells demonstrated a significant increase in platelet-activating factor (PAF) production when stimulated with thrombin [1 IU/ml, 10 min, 4,330 ± 555 vs. 15,227 ± 1,043 disintegrations per minute (dpm), P < 0.01] or TNF-α (10 ng/ml, 2 h, 16,532 ± 538 dpm, P < 0.01). Adherence of E0771 cells to WT endothelial cells was increased by thrombin (4.8 ± 0.3% vs. 70.9 ± 6.3, P < 0.01) or TNF-α (60.5 ± 4.3, P < 0.01). These responses were blocked by pretreatment with the iPLA(2)β-selective inhibitor (S)-bromoenol lactone and absent in lung endothelial cells from iPLA(2)β-KO mice. These data indicate that endothelial cell iPLA(2)β is responsible for PAF production and adherence of E0771 cells and may play a role in cancer cell migration to distal locations.

Imaging decreased brain docosahexaenoic acid metabolism and signaling in iPLA2β (VIA)-deficient mice

Ca(2+)-independent phospholipase A(2)β (iPLA(2)β) selectively hydrolyzes docosahexaenoic acid (DHA, 22:6n-3) in vitro from phospholipid. Mutations in the PLA2G6 gene encoding this enzyme occur in patients with idiopathic neurodegeneration plus brain iron accumulation and dystonia-parkinsonism without iron accumulation, whereas mice lacking PLA2G6 show neurological dysfunction and neuropathology after 13 months. We hypothesized that brain DHA metabolism and signaling would be reduced in 4-month-old iPLA(2)β-deficient mice without overt neuropathology. Saline or the cholinergic muscarinic M(1,3,5) receptor agonist arecoline (30 mg/kg) was administered to unanesthetized iPLA(2)β(-/-), iPLA(2)β(+/-), and iPLA(2)β(+/+) mice, and [1-(14)C]DHA was infused intravenously. DHA incorporation coefficients k* and rates J(in), representing DHA metabolism, were determined using quantitative autoradiography in 81 brain regions. iPLA(2)β(-/-) or iPLA(2)β(+/-) compared with iPLA(2)β(+/+) mice showed widespread and significant baseline reductions in k* and J(in) for DHA. Arecoline increased both parameters in brain regions of iPLA(2)β(+/+) mice but quantitatively less so in iPLA(2)β(-/-) and iPLA(2)β(+/-) mice. Consistent with iPLA(2)β's reported ability to selectively hydrolyze DHA from phospholipid in vitro, iPLA(2)β deficiency reduces brain DHA metabolism and signaling in vivo at baseline and following M(1,3,5) receptor activation. Positron emission tomography might be used to image disturbed brain DHA metabolism in patients with PLA2G6 mutations.

Genetic ablation of calcium-independent phospholipase A2β causes hypercontractility and markedly attenuates endothelium-dependent relaxation to acetylcholine

Activation of phospholipases leads to the release of arachidonic acid and lysophospholipids that play prominent roles in regulating vasomotor tone. To identify the role of calcium-independent phospholipase A(2)beta (iPLA(2)beta) in vasomotor function, we measured vascular responses to phenylephrine (PE) and ACh in mesenteric arterioles from wild-type (WT; iPLA(2)beta(+/+)) mice and those lacking the beta-isoform (iPLA(2)beta(-/-)) both ex vivo and in vivo. Vessels isolated from iPLA(2)beta(-/-) mice demonstrated increased constriction to PE, despite lower basal smooth muscle calcium levels, and decreased vasodilation to ACh compared with iPLA(2)beta(+/+) mice. PE constriction resulted in initial intracellular calcium release with subsequent steady-state constriction that depended on extracellular calcium influx. Endothelial denudation had no effect on vessel tone or PE-induced constriction although the dilation to ACh was significantly reduced in iPLA(2)beta(+/+) vessels. In contrast, vessels from iPLA(2)beta(-/-) constricted by 54% after denudation, indicating smooth muscle hypercontractility. In vivo, blood pressure, resting vessel diameter, and constriction of mesenteric vessels to PE were not different in iPLA(2)beta(-/-) vessels compared with WT mouse vessels. However, relaxation after ACh administration in situ was attenuated, indicating an endothelial inability to induce dilation in response to ACh. In cultured endothelial cells, inhibition of iPLA(2)beta with (S)-(E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL) decreased endothelial nitric oxide synthase phosphorylation and reduced endothelial agonist-induced intracellular calcium release as well as extracellular calcium influx. We conclude that iPLA(2)beta is an important mediator of vascular relaxation and intracellular calcium homeostasis in both smooth muscle and endothelial cells and that ablation of iPLA(2)beta causes agonist-induced smooth muscle hypercontractility and reduced agonist-induced endothelial dilation.

Role of Calcium-independent Phospholipase A2β in High Glucose-induced Activation of RhoA, Rho Kinase, and CPI-17 in Cultured Vascular Smooth Muscle Cells and Vascular Smooth Muscle Hypercontractility in Diabetic Animals

Previous studies suggest that high glucose-induced RhoA/Rho kinase/CPI-17 activation is involved in diabetes-associated vascular smooth muscle hypercontractility. However, the upstream signaling that links high glucose and RhoA/Rho kinase/CPI-17 activation is unknown. Here we report that calcium-independent phospholipase A(2)beta (iPLA(2)beta) is required for high glucose-induced RhoA/Rho kinase/CPI-17 activation and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility. We demonstrate that high glucose increases iPLA(2)beta mRNA, protein, and iPLA(2) activity in a time-dependent manner. Protein kinase C is involved in high glucose-induced iPLA(2)beta protein up-regulation. Inhibiting iPLA(2)beta activity with bromoenol lactone or preventing its expression by genetic deletion abolishes high glucose-induced RhoA/Rho kinase/CPI-17 activation, and restoring expression of iPLA(2)beta in iPLA(2)beta-deficient cells also restores high glucose-induced CPI-17 phosphorylation. Pharmacological and genetic inhibition of 12/15-lipoxygenases has effects on high glucose-induced CPI-17 phosphorylation similar to iPLA(2)beta inhibition. Moreover, increases in iPLA(2) activity and iPLA(2)beta protein expression are also observed in both type 1 and type 2 diabetic vasculature. Pharmacological and genetic inhibition of iPLA(2)beta, but not iPLA(2)gamma, diminishes diabetes-associated vascular smooth muscle hypercontractility. In summary, our results reveal a novel mechanism by which high glucose-induced, protein kinase C-mediated iPLA(2)beta up-regulation activates the RhoA/Rho kinase/CPI-17 via 12/15-lipoxygenases and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility.

Smooth Muscle Cell Arachidonic Acid Release, Migration, and Proliferation Are Markedly Attenuated in Mice Null for Calcium-independent Phospholipase A2β

Pharmacologic evidence suggests that the lipid products generated by one or more calcium-independent phospholipases A(2) (iPLA(2)s) participate in the regulation of vascular tone through smooth muscle cell (SMC) Ca(2+) signaling and the release of arachidonic acid. However, the recent identification of new members of the iPLA(2) family, each inhibitable by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one, has rendered definitive identification of the specific enzyme(s) mediating these processes difficult. Accordingly, we used iPLA(2)beta(-/-) mice to demonstrate that iPLA(2)beta is responsible for the majority of thapsigargin and ionophore (A23187)-induced arachidonic acid release from SMCs. Both thapsigargin and A23187 stimulated robust [(3)H]arachidonate (AA) release from wild-type aortic SMCs that was dramatically attenuated in iPLA(2)beta(-/-) mice (>80% reduction at 5 min; p < 0.01). Moreover, iPLA(2)beta(-/-) mice displayed defects in SMC Ca(2+) homeostasis and decreased SMC migration and proliferation in a model of vascular injury. Ca(2+)-store depletion resulted in the rapid entry of external Ca(2+) into wild-type aortic SMCs that was significantly slower in iPLA(2)beta-null cells (p < 0.01). Furthermore, SMCs from iPLA(2)beta-null mesenteric arterial explants demonstrated decreased proliferation and migration. The defects in migration and proliferation in iPLA(2)beta-null SMCs were restored by 2 mum AA. Remarkably, the cyclooxygenase-2-specific inhibitor, NS-398, prevented AA-induced rescue of SMC migration and proliferation in iPLA(2)beta(-/-) mice. Moreover, PGE(2) alone rescued proliferation and migration in iPLA(2)beta(-/-) mice. We conclude that iPLA(2)beta is an important mediator of AA release and prostaglandin E(2) production in SMCs, modulating vascular tone, cellular signaling, proliferation, and migration.

Highly Selective Hydrolysis of Fatty Acyl-CoAs by Calcium-independent Phospholipase A2β: ENZYME AUTOACYLATION AND ACYL-CoA-MEDIATED REVERSAL OF CALMODULIN INHIBITION OF PHOSPHOLIPASE A2 ACTIVITY

Calcium-independent phospholipase A2beta (iPLA2beta) participates in numerous diverse cellular processes, such as arachidonic acid release, insulin secretion, calcium signaling, and apoptosis. Herein, we demonstrate the highly selective iPLA2beta-catalyzed hydrolysis of saturated long-chain fatty acyl-CoAs (palmitoyl-CoA approximately myristoyl-CoA >> stearoyl-CoA >> oleoyl-CoA approximately = arachidonoyl-CoA) present either as monomers in solution or guests in host membrane bilayers. Site-directed mutagenesis of the iPLA2beta catalytic serine (S465A) completely abolished acyl-CoA thioesterase activity, demonstrating that Ser-465 catalyzes both phospholipid and acyl-CoA hydrolysis. Remarkably, incubation of iPLA2beta with oleoyl-CoA, but not other long-chain acyl-CoAs, resulted in robust stoichiometric covalent acylation of the enzyme. Moreover, S465A mutagenesis or pretreatment of wild-type iPLA2beta with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one unexpectedly increased acylation of the enzyme, indicating the presence of a second reactive nucleophilic residue that participates in the formation of the fatty acyl-iPLA2beta adduct. Radiolabeling of intact Sf9 cells expressing iPLA2beta with [3H]oleic acid demonstrated oleoylation of the membrane-associated enzyme. Partial trypsinolysis of oleoylated iPLA2beta and matrix-assisted laser desorption ionization mass spectrometry analysis localized the acylation site to a hydrophobic 25-kDa fragment (residues approximately 400-600) spanning the active site to the calmodulin binding domain. Intriguingly, calmodulin-Ca2+ blocked acylation of iPLA2beta by oleoyl-CoA. Remarkably, the addition of low micromolar concentrations (5 microM) of oleoyl-CoA resulted in reversal of calmodulin-mediated inhibition of iPLA2 beta phospholipase A2 activity. These results collectively identify the molecular species-specific acyl-CoA thioesterase activity of iPLA2beta, demonstrate the presence of a second active site that mediates iPLA2beta autoacylation, and identify long-chain acyl-CoAs as potential candidates mediating calcium influx factor activity.