A selective peroxisome proliferator-activated receptor δ agonist PYPEP suppresses atherosclerosis in association with improvement of the serum lipoprotein profiles in human apolipoprotein B100 and cholesteryl ester transfer protein double transgenic mice.

OBJECTIVE: Although peroxisome proliferator-activated receptor (PPAR) δ agonists have been shown to improve the serum lipoprotein profiles in humans, the impact of the changes in these lipoprotein profiles on atherosclerosis remains to be elucidated. The aim of this study was to investigate the relationship between the selective PPARδ agonist-induced alterations of serum lipoprotein profiles and the development of atherosclerosis in human apolipoprotein B100 and cholesterol ester transfer protein double transgenic (hApoB100/hCETP-dTg) mice with human-like hypercholesterolemic dyslipidemia. METHODS: hApoB100/hCETP-dTg mice fed an atherogenic diet received a novel PPARδ agonist (PYPEP) or vehicle for 18 weeks, followed by evaluation of atherosclerosis. Serum samples were collected during the treatment period at least at 3-week intervals to determine the lipoprotein levels and the levels of an inflammatory marker, macrophage chemotactic protein-1 (MCP-1), and to analyze the lipoprotein profile by fast protein liquid chromatography. The cholesterol efflux capacity of high-density lipoprotein (HDL) was examined using [(3)H]-cholesterol labeled macrophages. RESULTS: Compared with vehicle treatment, PYPEP treatment caused increases in the serum levels of HDL cholesterol and apolipoprotein A-I (ApoA-I), as well as reductions in the serum non-HDL cholesterol and MCP-1 levels. The HDL fraction from the PYPEP-treated group maintained its cholesterol efflux capacity and showed an increased population of smaller HDL particles. PYPEP substantially suppressed atherosclerotic lesion progression, and the lesion areas had significant correlations with non-HDL cholesterol, HDL cholesterol, ApoA-I and MCP-1 by Pearson's correlation analysis. A multiple regression analysis revealed that non-HDL cholesterol and ApoA-I were significantly associated with the atherosclerotic lesion area. CONCLUSION: A novel PPARδ agonist, PYPEP, suppressed atherosclerotic lesion progression by improving the serum lipoprotein profiles, including increased levels of ApoA-I and functional HDL particles, as well as a reduced non-HDL cholesterol level, in hApoB100/hCETP-dTg mice with human-like hypercholesterolemic dyslipidemia.

Pharmacological evaluation of adipose dysfunction via 11ß-hydroxysteroid dehydrogenase type 1 in the development of diabetes in diet-induced obese mice with cortisone pellet implantation.

Signals from intracellular glucocorticoids (GCs) via 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) in adipose tissues have been reported to serve as amplifiers leading to deterioration of glucose metabolism associated with obesity. To elucidate adipose dysfunction via 11ß-HSD1 activation in the development of obesity-related diabetes, we established novel diabetic mice by implanting a cortisone pellet (CP) in diet-induced obesity (DIO) mice. Cortisone pellet-implanted DIO mice (DIO/CP mice) showed hyperglycemia, insulin resistance, hyperlipidemia, and ectopic fat accumulation, whereas cortisone pellet implantation in lean mice did not induce hyperglycemia. In DIO/CP mice, indexes of lipolysis such as plasma glycerol and nonesterified fatty acids (NEFAs) increased before hyperglycemia appeared. Furthermore, the adipose mRNA level of 11ß-HSD1 was up-regulated in DIO/CP mice compared with sham-operated DIO mice. RU486 (mifepristone, 11ß-[p-(dimethylamino)phenyl]-17ß-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one), a glucocorticoid receptor antagonist, decreased adipose mRNA levels of 11ß-HSD1 as well as adipose triglyceride lipase. RU486 also improved plasma NEFA, glycerol, and glucose levels in DIO/CP mice. These results demonstrate that lipolysis in adipose tissues caused by GC activation via 11ß-HSD1 serves as a trigger for diabetes with ectopic fat accumulation. Our findings also indicate the possibility of a vicious circle of GC signals via 11ß-HSD1 up-regulation in adipose tissues, contributing to deterioration of glucose metabolism to result in diabetes. Our DIO/CP mouse could be a suitable model of type 2 diabetes to evaluate adipose dysfunction via 11ß-HSD1.

Lack of phospholipase A2 receptor increases susceptibility to cardiac rupture after myocardial infarction.

RATIONALE: Recent evidence indicates that the biological effects of secretory phospholipase A2 (sPLA2) cannot be fully explained by its catalytic activity. A cell surface receptor for sPLA2 (PLA2 receptor 1 [PLA2R]) and its high-affinity ligands (including sPLA2-IB, sPLA2-IIE, and sPLA2-X) are expressed in the infarcted myocardium. OBJECTIVE: This study asked whether PLA2R might play a pathogenic role in myocardial infarction (MI) using mice lacking PLA2R (PLA2R(-/-)). METHODS AND RESULTS: MI was induced by permanent ligation of the left coronary artery. PLA2R(-/-) mice exhibited higher rates of cardiac rupture after MI compared with PLA2R wild-type (PLA2R(+/+)) mice (46% versus 21%, respectively; P=0.015). PLA2R(-/-) mice had a 31% decrease in collagen content and a 45% decrease in the number of α-smooth muscle actin-positive fibroblasts in the infarcted region compared with PLA2R(+/+) mice. PLA2R was primarily found in myofibroblasts in the infarcted region. PLA2R(-/-) myofibroblasts were impaired in collagen-dependent migration, proliferation, and activation of focal adhesion kinase in response to sPLA2-IB. Binding of sPLA2-IB to PLA2R promoted migration and proliferation of myofibroblasts through functional interaction with integrin ß1, independent of the catalytic activity of sPLA2-IB. In rescue experiments, the injection of PLA2R(+/+) myofibroblasts into the infarcted myocardium prevented post-MI cardiac rupture and reversed the decrease in collagen content in the infarcted region in PLA2R(-/-) mice. CONCLUSIONS: PLA2R deficiency increased the susceptibility to post-MI cardiac rupture through impaired healing of the infarcted region. This might be partly explained by a reduction in integrin ß1-mediated migratory and proliferative responses of PLA2R(-/-) myofibroblasts.

Deficiency of phospholipase A2 receptor exacerbates ovalbumin-induced lung inflammation.

Secretory phospholipase A2 (sPLA2) plays a critical role in the genesis of lung inflammation through proinflammatory eicosanoids. A previous in vitro experiment showed a possible role of cell surface receptor for sPLA2 (PLA2R) in the clearance of extracellular sPLA2. PLA2R and groups IB and X sPLA2 are expressed in the lung. This study examined a pathogenic role of PLA2R in airway inflammation using PLA2R-deficient (PLA2R(-/-)) mice. Airway inflammation was induced by immunosensitization with OVA. Compared with wild-type (PLA2R(+/+)) mice, PLA2R(-/-) mice had a significantly greater infiltration of inflammatory cells around the airways, higher levels of groups IB and X sPLA2, eicosanoids, and Th2 cytokines, and higher numbers of eosinophils and neutrophils in bronchoalveolar lavage fluid after OVA treatment. In PLA2R(-/-) mice, intratracheally instilled [(125)I]-labeled sPLA2-IB was cleared much more slowly from bronchoalveolar lavage fluid compared with PLA2R(+/+) mice. The degradation of the instilled [(125)I]-labeled sPLA2-IB, as assessed by trichloroacetic acid-soluble radioactivity in bronchoalveolar lavage fluid after instillation, was lower in PLA2R(-/-) mice than in PLA2R(+/+) mice. In conclusion, PLA2R deficiency increased sPLA2-IB and -X levels in the lung through their impaired clearance from the lung, leading to exaggeration of lung inflammation induced by OVA treatment in a murine model.

Selective CB2 agonists with anti-pruritic activity: discovery of potent and orally available bicyclic 2-pyridones.

The CB2 receptor has emerged as a potential target for the treatment of pruritus as well as pain without CB1-mediated side effects. We previously identified 2-pyridone derivatives 1 and 2 as potent CB2 agonists; however, this series of compounds was found to have unacceptable pharmacokinetic profiles with no significant effect in vivo. To improve these profiles, we performed further structural optimization of 1 and 2, which led to the discovery of bicyclic 2-pyridone 18e with improved CB2 affinity and selectivity over CB1. In a mouse pruritus model, 18e inhibited compound 48/80 induced scratching behavior at a dose of 100 mg/kg. In addition, the docking model of 18e with an active-state CB2 homology model indicated the structural basis of its high affinity and selectivity over CB1.

Design, synthesis, and binding mode prediction of 2-pyridone-based selective CB2 receptor agonists.

Selective CB2 agonists have the potential for treating pain without central CB1-mediated adverse effects. Screening efforts identified 1,2-dihydro-3-isoquinolone 1; however, this compound has the drawbacks of being difficult to synthesize with two asymmetric carbons on an isoquinolone scaffold and of having a highly lipophilic physicochemical property. To address these two major problems, we designed the 2-pyridone-based lead 15a, which showed moderate affinity for CB2. Optimization of 15a led to identification of 39f with high affinity for CB2 and selectivity over CB1. Prediction of the binding mode of 39f in complex with an active-state CB2 homology model provided structural insights into its high affinity for CB2.

Differences between group X and group V secretory phospholipase A(2) in lipolytic modification of lipoproteins.

Secretory phospholipases A(2) (sPLA(2)s) are a diverse family of low molecular mass enzymes (13-18 kDa) that hydrolyze the sn-2 fatty acid ester bond of glycerophospholipids to produce free fatty acids and lysophospholipids. We have previously shown that group X sPLA(2) (sPLA(2)-X) had a strong hydrolyzing activity toward phosphatidylcholine in low-density lipoprotein (LDL) linked to the formation of lipid droplets in the cytoplasm of macrophages. Here, we show that group V sPLA(2) (sPLA(2)-V) can also cause the lipolysis of LDL, but its action differs remarkably from that of sPLA(2)-X in several respects. Although sPLA(2)-V released almost the same amount of fatty acids from LDL, it released more linoleic acid and less arachidonic acid than sPLA(2)-X. In addition, the requirement of Ca(2+) for the lipolysis of LDL was about 10-fold higher for sPLA(2)-V than sPLA(2)-X. In fact, the release of fatty acids from human serum was hardly detectable upon incubation with sPLA(2)-V in the presence of sodium citrate, which contrasted with the potent response to sPLA(2)-X. Moreover, sPLA(2)-X, but not sPLA(2)-V, was found to specifically interact with LDL among the serum proteins, as assessed by gel-filtration chromatography as well as sandwich enzyme-immunosorbent assay using anti-sPLA(2)-X and anti-apoB antibodies. Surface plasmon resonance studies have revealed that sPLA2-X can bind to LDL with high-affinity (K(d) = 3.1 nM) in the presence of Ca(2+). Selective interaction of sPLA(2)-X with LDL might be involved in the efficient hydrolysis of cell surface or intracellular phospholipids during foam cell formation.

Group X secretory PLA2 in neutrophils plays a pathogenic role in abdominal aortic aneurysms in mice.

Group X secretory PLA(2) (sPLA(2)-X) is expressed in neutrophils and plays a role in the pathogenesis of neutrophil-mediated tissue inflammation and injury. This study tested the hypothesis that sPLA(2)-X in neutrophils may contribute to the pathogenesis of abdominal aortic aneurysms (AAA) using sPLA(2)-X(-/-) mice. AAA was created by application of CaCl(2) to external surface of aorta. As a result, the aortas of sPLA(2)-X(-/-) mice had smaller diameters (percent increase from baseline; 24.8 ± 3.5% vs. 49.9 ± 9.1%, respectively; P < 0.01), a reduced grade of elastin degradation, and lower activities of elastase and gelatinase (26% and 19% lower, respectively) after CaCl(2) treatment compared with sPLA(2)-X(+/+) mice. In sPLA(2)-X(+/+) mice, immunofluorescence microscopic images showed that the immunoreactivity of sPLA(2)-X was detected only in neutrophils within aortic walls 3 days, 1, 2, and 6 wk after CaCl(2) treatment, whereas the immunoreactivity was not detected in macrophages or mast cells in aortic walls. sPLA(2)-X immunoreactivity also was colocalized in cells expressing matrix metalloproteinase (MMP)-9. Neutrophils isolated from sPLA(2)-X(-/-) mice had lower activities of elastase, gelatinase, and MMP-9 in response to stimuli compared with sPLA(2)-X(+/+) mice. The attenuated release of elastase and gelatinase from sPLA(2)-X(-/-) neutrophils was reversed by exogenous addition of mouse sPLA(2)-X protein. The adoptive transfer of sPLA(2)-X(+/+) neutrophils days 0 and 3 after CaCl(2) treatment reversed aortic diameters and elastin degradation grades in the lethally irradiated sPLA(2)-X(+/+) mice reconstituted with sPLA(2)-X(-/-) bone marrow to an extent similar to that seen in sPLA(2)-X(+/+) mice. In conclusion, sPLA(2)-X in neutrophils plays a pathogenic role in AAA in a mice model.

Physiological roles of group X-secreted phospholipase A2 in reproduction, gastrointestinal phospholipid digestion, and neuronal function.

Although the secreted phospholipase A(2) (sPLA(2)) family has been generally thought to participate in pathologic events such as inflammation and atherosclerosis, relatively high and constitutive expression of group X sPLA(2) (sPLA(2)-X) in restricted sites such as reproductive organs, the gastrointestinal tract, and peripheral neurons raises a question as to the roles played by this enzyme in the physiology of reproduction, digestion, and the nervous system. Herein we used mice with gene disruption or transgenic overexpression of sPLA(2)-X to clarify the homeostatic functions of this enzyme at these locations. Our results suggest that sPLA(2)-X regulates 1) the fertility of spermatozoa, not oocytes, beyond the step of flagellar motility, 2) gastrointestinal phospholipid digestion, perturbation of which is eventually linked to delayed onset of a lean phenotype with reduced adiposity, decreased plasma leptin, and improved muscle insulin tolerance, and 3) neuritogenesis of dorsal root ganglia and the duration of peripheral pain nociception. Thus, besides its inflammatory action proposed previously, sPLA(2)-X participates in physiologic processes including male fertility, gastrointestinal phospholipid digestion linked to adiposity, and neuronal outgrowth and sensing.

Hair follicular expression and function of group X secreted phospholipase A2 in mouse skin.

Although perturbed lipid metabolism can often lead to skin abnormality, the role of phospholipase A(2) (PLA(2)) in skin homeostasis is poorly understood. In the present study we found that group X-secreted PLA(2) (sPLA(2)-X) was expressed in the outermost epithelium of hair follicles in synchrony with the anagen phase of hair cycling. Transgenic mice overexpressing sPLA(2)-X (PLA2G10-Tg) displayed alopecia, which was accompanied by hair follicle distortion with reduced expression of genes related to hair development, during a postnatal hair cycle. Additionally, the epidermis and sebaceous glands of PLA2G10-Tg skin were hyperplasic. Proteolytic activation of sPLA(2)-X in PLA2G10-Tg skin was accompanied by preferential hydrolysis of phosphatidylethanolamine species with polyunsaturated fatty acids as well as elevated production of some if not all eicosanoids. Importantly, the skin of Pla2g10-deficient mice had abnormal hair follicles with noticeable reduction in a subset of hair genes, a hypoplasic outer root sheath, a reduced number of melanin granules, and unexpected up-regulation of prostanoid synthesis. Collectively, our study highlights the spatiotemporal expression of sPLA(2)-X in hair follicles, the presence of skin-specific machinery leading to sPLA(2)-X activation, a functional link of sPLA(2)-X with hair follicle homeostasis, and compartmentalization of the prostanoid pathway in hair follicles and epidermis.

Irbesartan enhances GLUT4 translocation and glucose transport in skeletal muscle cells.

Irbesartan, an angiotensin II type 1 receptor blocker has been reported to alleviate metabolic disorder in animal studies and human clinical trials. Although this effect may be related to the ability of irbesartan to serve as a partial agonist for the peroxisome proliferator-activated receptor (PPAR)-γ, the target tissues on which irbesartan acts remain poorly defined. As muscle glucose transport plays a major role in maintaining systemic glucose homeostasis, we investigated the effect of irbesartan on glucose uptake in skeletal muscle cells. In C2C12 myotubes, 24-h treatment with irbesartan significantly promoted both basal and insulin-stimulated glucose transport. In L6-GLUT4myc myoblasts, irbesartan caused a significant increase in glucose transport and GLUT4 translocation to the cell surface in a concentration-dependent manner. Valsartan, another angiotensin II type 1 receptor blocker had no effect on either glucose uptake or GLUT4 translocation, implying that these actions on glucose transport are independent of angiotensin II receptor blockade. Moreover, irbesartan exerted these effects in an additive manner with insulin, but not with acute treatment for 3 h, suggesting that they may require the synthesis of new proteins. Finally, in insulin-resistant Zucker fatty rat, irbesartan (50 mg/kg/day for 3 weeks) significantly ameliorated insulin resistance without increasing weight gain. We conclude that irbesartan has a direct action, which can be additive to insulin, of promoting glucose transport in skeletal muscle. This may be beneficial for ameliorating obesity-related glucose homeostasis derangement.

Identification of glycosylated exendin-4 analogue with prolonged blood glucose-lowering activity through glycosylation scanning substitution.

Exendin-4, a glucagon-like peptide 1 receptor agonist, is a potent therapeutic xenopeptide hormone for the treatment of type 2 diabetes. In order to further improve in vivo activity, we examined the introduction of sialyl N-acetyllactosamine (sialyl LacNAc) to exendin-4. The glycosylated analogue having sialyl LacNAc at position 28 was found to have improved in vivo activity with prolonged glucose-lowering activity.

Gly80Ser polymorphism of phospholipase A2-IID is associated with cytokine inducibility in A549 cells.

BACKGROUND: The Gly80Ser polymorphism in phospholipase A2-IID (PLA2G2D, NCBI SNP reference: rs584367) is associated with a loss in body weight in patients with chronic obstructive pulmonary disease (COPD). The T allele missense mutation results in the 80th amino acid of the PLA2G2D protein changing from a glycine (Gly; C allele) to a serine (Ser; T allele). COPD patients carrying Ser lose a significant amount of weight compared with those carrying Gly. The mechanism for this weight loss following carriage of this Ser allele has not been clarified. OBJECTIVES: We aimed to evaluate whether this allelic change alters PLA2 enzymatic activity and/or pro-inflammatory cytokine inducibility. METHODS: A549 cells (a human pulmonary epithelial cell line) were transfected with PLA2G2D-Gly or PLA2G2D-Ser. We evaluated PLA2 activity and cytokine expressions in these cells. RESULTS: The enzymatic activity of sPLA2 in A549-PLA2G2D-Ser cells did not differ from the A549-PLA2G2D-Gly cells. A549-PLA2G2D-Ser cells spontaneously produced higher levels of interleukin (IL)-6 and IL-8 than A549-PLA2G2D-Gly cells. Upon tumor necrosis factor-alpha stimulation, IL-6 and IL-8 mRNA and protein levels in A549-PLA2G2D-Ser cells were elevated compared with those of A549-PLA2G2D-Gly cells. Upon hydrogen peroxide stimulation, IL-8 mRNA and protein levels in A549-PLA2G2D-Ser cells were higher than those of A549-PLA2G2D-Gly cells. CONCLUSIONS: PLA2G2D-Ser enhances the expression of IL-6 and IL-8 compared with PLA2G2D-Gly. This enhanced cytokine expression observed with the allelic change in PLA2G2D may be associated with the body weight loss seen in COPD patients.

Chemoenzymatic synthesis of glycosylated glucagon-like peptide 1: effect of glycosylation on proteolytic resistance and in vivo blood glucose-lowering activity.

Glucagon-like peptide 1 (7-36) amide (GLP-1) has been attracting considerable attention as a therapeutic agent for the treatment of type 2 diabetes. In this study, we applied a glycoengineering strategy to GLP-1 to improve its proteolytic stability and in vivo blood glucose-lowering activity. Glycosylated analogues with N-acetylglucosamine (GlcNAc), N-acetyllactosamine (LacNAc), and alpha2,6-sialyl N-acetyllactosamine (sialyl LacNAc) were prepared by chemoenzymatic approaches. We assessed the receptor binding affinity and cAMP production activity in vitro, the proteolytic resistance against dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase (NEP) 24.11, and the blood glucose-lowering activity in diabetic db/db mice. Addition of sialyl LacNAc to GLP-1 greatly improved stability against DPP-IV and NEP 24.11 as compared to the native type. Also, the sialyl LacNAc moiety extended the blood glucose-lowering activity in vivo. Kinetic analysis of the degradation reactions suggested that the sialic acid component played an important role in decreasing the affinity of peptide to DPP-IV. In addition, the stability of GLP-1 against both DPP-IV and NEP24.11 incrementally improved with an increase in the content of sialyl LacNAc in the peptide. The di- and triglycosylated analogues with sialyl LacNAc showed greatly prolonged blood glucose-lowering activity of up to 5 h after administration (100 nmol/kg), although native GLP-1 showed only a brief duration. This study is the first attempt to thoroughly examine the effect of glycosylation on proteolytic resistance by using synthetic glycopeptides having homogeneous glycoforms. This information should be useful for the design of glycosylated analogues of other bioactive peptides as desirable pharmaceuticals.

Reduction in myocardial ischemia/reperfusion injury in group X secretory phospholipase A2-deficient mice.

BACKGROUND: Group X secretory phospholipase A(2) (sPLA(2)-X) has the most potent hydrolyzing activity toward phosphatidylcholine and elicits a marked release of arachidonic acid among several types of sPLA(2). sPLA(2)-X is expressed in neutrophils, but its pathogenic role remains unclear. METHODS AND RESULTS: We generated mice that lack sPLA(2)-X and studied their response to myocardial ischemia/reperfusion. The sPLA(2)-X(-/-) mice had a significant reduction in myocardial infarct size and a decrease in myocardial myeloperoxidase activity compared with sPLA(2)-X(+/+) mice. Myocardial infarct size was also significantly reduced in lethally irradiated sPLA(2)-X(+/+) mice reconstituted with sPLA(2)-X(-/-) bone marrow compared with sPLA(2)-X(+/+) bone marrow. The extent of myocardial ischemia/reperfusion injury was comparable between sPLA(2)-X(-/-) and sPLA(2)-X(+/+) mice in Langendorff experiments using isolated hearts and blood-free perfusion buffer, supporting a potential role of sPLA(2)-X in blood in myocardial ischemia/reperfusion injury. In the infarcted myocardium of sPLA(2)-X(+/+) mice, sPLA(2)-X was released from neutrophils but not myocardial tissues and platelets and was undetectable in the peripheral serum. The sPLA(2)-X(-/-) mice had lower accumulation of neutrophils in ischemic myocardium, and the isolated sPLA(2)-X(-/-) neutrophils had lower release of arachidonic acid and attenuated cytotoxic activities including respiratory burst compared with sPLA(2)-X(+/+) neutrophils. The attenuated functions of sPLA(2)-X(-/-) neutrophils were reversible by the exogenous addition of sPLA(2)-X protein. Furthermore, administration of a sPLA(2) inhibitor reduced myocardial infarct size and suppressed the cytotoxic activity of sPLA(2)-X(+/+) neutrophils. CONCLUSIONS: Myocardial ischemia/reperfusion injury was attenuated in sPLA(2)-X(-/-) mice partly through the suppression of neutrophil cytotoxic activities.

2-Arylimino-5,6-dihydro-4H-1,3-thiazines as a new class of cannabinoid receptor agonists. Part 1: discovery of CB2 receptor selective compounds.

2-Arylimino-5,6-dihydro-4H-1,3-thiazines have been identified as a novel class of cannabinoid agonists. A lead structure with moderate activity was discovered through a high throughput screening assay. Structure-activity relationships led to the discovery of potent agonists of CB(2) receptor. The most potent compound 13 displays K(i) values of >5000 and 9 nM to CB(1) and CB(2) receptors, respectively.

2-Arylimino-5,6-dihydro-4H-1,3-thiazines as a new class of cannabinoid receptor agonists. Part 2: orally bioavailable compounds.

Structure-activity relationships and efforts to optimize the pharmacokinetic profile of a class of 2-arylimino-5,6-dihydro-4H-1,3-thiazines as cannabinoid receptor agonists are described. Among the compounds examined, compound 14 showed potent affinity and high selectivity for CB2, and compound 23 showed potent affinities against CB1 and CB2. These compounds displayed oral bioavailability.

Group X secretory phospholipase A2 can induce arachidonic acid release and eicosanoid production without activation of cytosolic phospholipase A2 alpha.

Group X secretory phospholipase A2 (sPLA2-X) and cytosolic phospholipase A2 alpha (cPLA2alpha) are involved in the release of arachidonic acid (AA) from membrane phospholipids linked to the eicosanoid production in various pathological states. Recent studies have indicated the presence of various types of cross-talk between sPLA2s and cPLA2alpha resulting in effective AA release. Here we examined the dependence of sPLA2-X-induced potent AA release on the cPLA2alpha activation by using specific cPLA2alpha or sPLA2 inhibitors as well as cPLA2alpha-deficient mice. We found that Pyrrophenone, a cPLA2alpha-specific inhibitor, did not suppress the sPLA2-X-induced potent AA release and prostaglandin E2 formation in mouse spleen cells. Furthermore, the amount of AA released by sPLA2-X from spleen cells was not significantly altered by cPLA2alpha deficiency. These results suggest that sPLA2-X induces potent AA release without activation of cPLA2a, which might be relevant to eicosanoid production in some pathological states where cPLA2a is not activated.

Mammalian phospholipase A2: phospholipase A2 receptor.

The phospholipase A2 receptor (PLA2R) is a type I transmembrane glycoprotein related to the C-type animal lectin family such as the mannose receptor. PLA2R regulates a variety of biological responses elicited by secretory phospholipase A2s (sPLA2s). Group IB sPLA2 acts as an endogenous ligand to induce cell proliferation and lipid mediator production. Analysis of PLA2R-deficient mice suggested a potential role of the sPLA2-IB/PLA2R pathway in the production of proinflammatory cytokines during endotoxic shock. PLA2R is also involved in the clearance of sPLA2s, especially group X sPLA2, to protect their exaggerated reactions by potent enzymatic activities. In circulation, the soluble form of PLA2R is constitutively present as an endogenous inhibitor for mammalian sPLA2s.

Novel binding sites of 15-deoxy-Delta12,14-prostaglandin J2 in plasma membranes from primary rat cortical neurons.

15-Deoxy-Delta12,14-prostaglandin J2 (15d-Delta12,14-PGJ2) is an endogenous ligand for a nuclear peroxysome proliferator activated receptor-gamma (PPAR). We found novel binding sites of 15d-Delta12,14-PGJ2 in the neuronal plasma membranes of the cerebral cortex. The binding sites of [3H]15d-Delta12,14-PGJ2 were displaced by 15d-Delta12,14-PGJ2 with a half-maximal concentration of 1.6 microM. PGD2 and its metabolites also inhibited the binding of [3H]15d-Delta12,14-PGJ2. Affinities for the novel binding sites were 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. Other eicosanoids and PPAR agonists did not alter the binding of [3H]15d-Delta12,14-PGJ2. In primary cultures of rat cortical neurons, we examined the pathophysiologic roles of the novel binding sites. 15d-Delta12,14-PGJ2 triggered neuronal cell death in a concentration-dependent manner, with a half-maximal concentration of 1.1 microM. The neurotoxic potency of PGD2 and its metabolites was also 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. The morphologic and ultrastructural characteristics of 15d-Delta12,14-PGJ2-induced neuronal cell death were apoptotic, as evidenced by condensed chromatin and fragmented DNA. On the other hand, we detected little neurotoxicity of other eicosanoids and PPAR agonists. In conclusion, we demonstrated that novel binding sites of 15d-Delta12,14-PGJ2 exist in the plasma membrane. The present study suggests that the novel binding sites might be involved in 15d-Delta12,14-PGJ2-induced neuronal apoptosis.