Arachidonic Acid Release Research Articles

A rapid and adaptable lipidomics method for quantitative UPLC-mass spectrometric analysis of phosphatidylethanolamine and phosphatidylcholine in vitro, and in cells.

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are highly prevalent phospholipids in mammalian membranes. There are currently no methods for detection of minute levels of these phospholipids or simultaneously with products of the utilization of these phospholipid substrates by phospholipase A2 (PLA2) enzymes. To examine the substrate utilization of PE and PC by PLA2, we developed a method to accurately detect and measure specific forms of PE and PC as low as 50 femtomoles. Validation of this method consisted of an enzymatic assay to monitor docosahexaenoic acid and arachidonic acid release from the hydrolysis of PE and PC by group IV phospholipase A2 (cPLA2α) coupled to the generation of lyso-PE (LPE) and lyso-PC (LPC). In addition, the PE and PC profiles of RAW 264.7 macrophages were monitored with zymosan/lipopolysaccharide-treatment. Finally, genetic validation for the specificity of the method consisted of the downregulation of two biosynthetic enzymes responsible for the production of PE and PC, choline kinase A (CHKA) and ethanolamine kinase 1 (ETNK1). This new UPLC ESI-MS/MS method provides accurate and highly sensitive detection of PE and PC species containing AA and DHA allowing for the specific examination of the substrate utilization of these phospholipids by PLA2 in vitro and in cells.

CPLA2α-/- sympathetic neurons exhibit increased membrane excitability and loss of N-Type Ca2+ current inhibition by M1 muscarinic receptor signaling.

Group IVa cytosolic phospholipase A2 (cPLA2α) mediates GPCR-stimulated arachidonic acid (AA) release from phosphatidylinositol 4,5-bisphosphate (PIP2) located in plasma membranes. We previously found in superior cervical ganglion (SCG) neurons that PLA2 activity is required for voltage-independent N-type Ca2+ (N-) current inhibition by M1 muscarinic receptors (M1Rs). These findings are at odds with an alternative model, previously observed for M-current inhibition, where PIP2 dissociation from channels and subsequent metabolism by phospholipase C suffices for current inhibition. To resolve cPLA2α’s importance, we have investigated its role in mediating voltage-independent N-current inhibition (~40%) that follows application of the muscarinic agonist oxotremorine-M (Oxo-M). Preincubation with different cPLA2α antagonists or dialyzing cPLA2α antibodies into cells minimized N-current inhibition by Oxo-M, whereas antibodies to Ca2+-independent PLA2 had no effect. Taking a genetic approach, we found that SCG neurons from cPLA2α-/- mice exhibited little N-current inhibition by Oxo-M, confirming a role for cPLA2α. In contrast, cPLA2α antibodies or the absence of cPLA2α had no effect on voltage-dependent N-current inhibition by M2/M4Rs or on M-current inhibition by M1Rs. These findings document divergent M1R signaling mediating M-current and voltage-independent N-current inhibition. Moreover, these differences suggest that cPLA2α acts locally to metabolize PIP2 intimately associated with N- but not M-channels. To determine cPLA2α’s functional importance more globally, we examined action potential firing of cPLA2α+/+ and cPLA2α-/- SCG neurons, and found decreased latency to first firing and interspike interval resulting in a doubling of firing frequency in cPLA2α-/- neurons. These unanticipated findings identify cPLA2α as a tonic regulator of neuronal membrane excitability.

Digoxin and Platelet Activation in Patients With Atrial Fibrillation: In Vivo and In Vitro Study.

BackgroundDigoxin use was shown to be associated with an increased risk of cardiovascular events in atrial fibrillation (AF). We hypothesized that digoxin may affect cardiovascular risk by increasing platelet activation.Methods and ResultsPost hoc analysis of a prospective study of anticoagulated patients with AF. Patients were divided into 2 groups balanced for age, sex, and cardiovascular risk factors: digoxin users (n=132) and nonusers (n=388). Urinary excretion of 11‐dehydro‐thromboxane B2 (TxB2), a marker of platelet activation, and serum digoxin concentration (SDC) were measured. In vitro experiments were performed on platelets from healthy subjects and AF patients, which were incubated with scalar doses of digoxin (0.6–2.4 ng/mL) with or without prestimulation with a sub‐threshold of collagen. Median 11‐dehydro‐TxB2 was 105.0 (interquartile range, 60.0–190.0) ng/mg creatinine, and median SDC was 0.65 (interquartile range, 0.40–1.00) ng/mL. Urinary 11‐dehydro‐TxB2 and SDC were correlated (r s=0.350, P<0.001). Patients in the upper tertile of SDC showed higher 11‐dehydro‐TxB2 compared with non–digoxin users (P=0.019). In vitro study showed an increased basal platelet activation in patients with AF compared with healthy subjects. Digoxin (2.4 ng/mL) induced calcium mobilization, PAC‐1 (procaspase‐activating compound 1) and platelet aggregation in AF patients but not in healthy subjects. After pretreatment with a sub‐threshold of collagen, digoxin dose‐dependent induced calcium mobilization, arachidonic acid release, TxB2 biosynthesis, PAC‐1 and soluble platelet selectin expression, and platelet aggregation, which were inhibited by antibody against digoxin.ConclusionsWe found a significant in vivo correlation between SDC and platelet activation. Supratherapeutic SDC increased in vitro platelet aggregation via calcium‐related phospholipase A2 phosphorylation. Our findings may have clinical implications for AF patients treated with digoxin.

Caffeic Acid Diminishes the Production and Release of Thrombogenic Molecules in Human Platelets

Caffeic acid (CA) is a well-known to inhibit thrombogenic thromboxane A2 (TXA2) production, but there are no reports for the supply and usage of its precursor arachidonic acid (AA) and the release of serotonin, another thrombogenic molecule. In this study, we investigated the effect of CA on the phosphorylation of AA supply enzymes [cytosolic phospholipase A2α (cPLA2α), and phospholipase Cγ2 (PLCγ2)] and AA utilization enzymes [cyclooxygenase (COX)-1, TXA2 synthase (TXAS)] in collagen-activated human platelets. p38 MAPK phosphorylation associated with TXA2 production and c-Jun NH2-terminal kinase 1 (JNK1) phosphorylation associated with serotonin release were also determined. CA inhibited collagen-induced TXA2 production in a dose-dependent manner. It acted by counteracting the phosphorylation of AA release enzyme (PLCγ2) and AA utilization enzymes (COX-1 and TXAS) without affecting p38 MAPK and cPLA2α in collagenactivated human platelets. CA decreased collagen-released serotonin by inhibiting JNK1 phosphorylation. Therefore, our data demonstrate that CA might protect TXA2- and serotonin-mediated thrombogenesis.

Metabolic profiling of human plasma reveals the activation of 5-lipoxygenase in the acute attack of gouty arthritis.

Monosodium urate-induced inflammation plays a vital role in acute gout (AG). Inflammation is a multi-stage process involved in the acute release of arachidonic acid and its metabolites. However, the function of the metabolism of arachidonic acid and other polyunsaturated fatty acids in AG is not well understood. This study aimed to investigate the modification of polyunsaturated fatty acid metabolism by AG. Plasma samples from patients with an AG attack (n = 26) and gender-matched healthy controls (n = 26) were analysed by metabolic profiling of polyunsaturated fatty acids. The findings were further validated with a second cohort (n = 20 each group). The associated mechanisms were investigated in whole blood cells from the second cohort and neutrophils in vitro. Plasma metabolic profiling revealed a significant increase in leukotriene B4 (LTB4) for AG patients in both cohorts. The increase in plasma LTB4 was accounted for by the dynamic balance between the activation of 5-lipoxygenase and CYP4F3, the former mediating the biosynthesis of LTB4 and the latter mediating its metabolism. This was supported by significantly increased transcriptional levels of 5-lipoxygenase and CYP4F3 in whole blood cells from AG patients compared with those of controls, and the uric acid-caused dose-relevant and time-dependent activation of 5-lipoxygenase and CYP4F3 at the transcriptional and molecular levels in vitro. Increased LTB4 in AG patients is mainly due to activation of 5-lipoxygenase. 5-Lipoxygenase inhibition may be of therapeutic value clinically.

DMSO-induced drastic changes in cellular processes and epigenetic landscape in vitro

Classical FcεRI-induced mast cell (MC) activation causes synthesis of arachidonic acid (AA)–derived eicosanoids (leukotriene [LT] C4, prostaglandin [PG] D2, and thromboxane A2), which mediate vascular leak, bronchoconstriction, and effector cell chemotaxis. Little is known about the significance and regulation of eicosanoid generation in response to nonclassical MC activation mechanisms.We sought to determine the regulation and significance of MC-derived eicosanoids synthesized in response to IL-33, a cytokine critical to innate type 2 immunity.We used an ex vivo model of mouse bone marrow–derived mast cells and an IL-33–dependent in vivo model of aspirin-exacerbated respiratory disease (AERD).IL-33 potently liberates AA and elicits LTC4, PGD2, and thromboxane A2 production by bone marrow–derived mast cells. Unexpectedly, the constitutive function of COX-1 is required for IL-33 to activate group IVa cytosolic phospholipase A2 with consequent AA release for synthesis of all eicosanoids, including CysLTs. In contrast, COX-1 was dispensable for FcεRI-driven CysLT production. Inhibition of COX-1 prevented IL-33–induced phosphorylation of extracellular signal-related kinase, an upstream effector of cytosolic phospholipase A2, which was restored by exogenous PGH2, implying that the effects of COX-1 required its catalytic function. Administration of a COX-1–selective antagonist to mice completely prevented the generation of both PGD2 and LTC4 in a model of AERD in which MC activation is IL-33 driven.MC-intrinsic COX-1 amplifies IL-33–induced activation in the setting of innate type 2 immunity and might help explain the phenomenon of therapeutic desensitization to aspirin by nonselective COX inhibitors in patients with AERD.

Abstract P303: Inhibition of Group IV Cytosolic Phospholipase A 2 α by Cytochrome P450 1B1-Estradiol Derived Metabolite 2-Methoxyestradiol Protects Against Angiotensin II-Induced Hypertension in Female Mice

Previously we showed that the protection against angiotensin (Ang) II-induced hypertension and associated pathophysiological changes in female mice are mediated by cytochrome P450 (CYP) 1B1-estradiol (E2) generated metabolite 2-methoxyestradiol (2-ME). Also, we demonstrated that Ang II-induced hypertension is mediated by group IV cytosolic phospholipase A 2 α (cPLA 2 α) activation resulting in arachidonic acid (AA) release, and generation predominantly of eicosanoids with pro-hypertensive effects in male mice. This study was conducted to determine the contribution of cPLA 2 α/AA system, and its relationship to CYP1B1 in Ang II-induced hypertension in the female mice. Ang II infusion (700 ng/kg/min, s.c. micro-osmotic pump) for 14 days increased the systolic blood pressure (SBP), measured by tail-cuff in wild-type mice (cPLA 2 α +/+ ) but not in cPLA 2 α gene-disrupted mice (cPLA 2 α -/- ) (151±8 vs. 126±9 mmHg, P &lt;0.05, n=4-5). Ang II markedly increased SBP in ovariectomized (OVX) cPLA 2 α +/+ mice but not in OVX cPLA 2 α -/- mice (175±3 vs. 121±2 mmHg, P &lt;0.05, n=4-5). AA metabolism inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA, 50 mg/kg, i.p. every 3 rd day) attenuated ( P &lt;0.05) the Ang II-induced increase in SBP in intact (124±5 mmHg, n=5) and OVX cPLA 2 α +/+ mice (124±5 mmHg, n=4). E2 attenuated Ang II-induced increase in SBP in OVX Cyp1b1 +/+ mice, but not in OVX Cyp1b1 -/- mice (125±5 vs. 158±4 mmHg, P &lt;0.05, n=3-4). Ang II-induced increase in SBP was also attenuated by ETYA in the Cyp1b1 -/- mice (129±7 vs. 185±7 mmHg, P &lt;0.05, n=4). Ang II-induced increase in cPLA 2 α +/+ activity examined in the kidneys was inhibited in Cyp1b1 -/- mice treated with 2-ME. Antagonists of prostaglandin (PG) E2 receptors EP1 (SC19220) and EP3 (L-798106) (28 μg/g, s.c. every 2 nd day) minimized Ang II-induced increase in SBP in OVX cPLA 2 α +/+ mice (126±2 and 127±5 vs. 175±3 mmHg, respectively, n=5). These data suggest that CYP1B1-E2 generated metabolite 2-ME protects against Ang II-induced hypertension by inhibiting cPLA 2 α activity and production of AA-derived PGE2 that exerts pro-hypertensive effects by stimulating EP1 and EP3 receptors. Thus, 2-ME and the drugs that selectively block EP1 and EP3 receptors could be useful for the treatment of hypertension and its pathogenesis in females.

The inhibitory effect of saPLIγ, a snake sourced PLA2 inhibitor on carrageenan-induced inflammation in mice

SaPLIγ is a natural phospholipase A2 (PLA2) inhibitor, isolated from Sinonatrix annularis, that has been demonstrated to protect against envenomation by other venomous snakes. As snake venom PLA2s and mammalian secretory PLA2s are similar, saPLIγ is thought to have potential to alleviate inflammatory reactions in which PLA2s act as a key enzyme for arachidonic acid release. The aim of this study was to investigate the anti-inflammatory effects and mechanisms of action of saPLIγ in an animal model of carrageenan-induced acute inflammation. The results indicated that saPLIγ inhibited PLA2 subtypes extensively, especially IIA-PLA2, in a dose-dependent manner. Paw swelling in mice was reduced markedly by intraperitoneal saPLIγ 2.5 mg/kg, and the effect was significantly better than observed with dexamethasone at the same dose. Lower neutrophil infiltration and tissue edema was observed in the paws of saPLIγ-treated mice. Additionally, carrageenan-induced cyclooxygenase-2 (COX-2) and pro-inflammatory cytokines (TNFα and IL-1β) were also significantly down-regulated by saPLIγ in a dose-dependent manner. These results suggested that saPLIγ had effective anti-inflammatory effects in vivo, and these were produced by blocking mammalian IB, IIA, V and X sPLA2 subtypes.

Angiotensin (1–7) does not interact directly with MAS1, but can potently antagonize signaling from the AT1 receptor

Angiotensin (1–7) has been reported to be a ligand for the GPCR MAS1. Small molecule MAS1 modulators have also been recently characterized. Aside from convincing evidence for MAS1 activation of Gq signaling, little is known about MAS1 mediated signaling pathways initiated by these ligands, especially Ang (1–7). We performed a comprehensive characterization of recombinant MAS1 signaling induced by Ang (1–7) and small molecule ligands through numerous G protein-dependent and independent pathways, and in a signaling pathway agnostic approach. We find that small molecule ligands modulate numerous G protein-dependent and independent pathways through MAS1, including Gq and Gi pathways, GTPγS binding, β-arrestin recruitment, Erk1/2 and Akt phosphorylation, arachidonic acid release, and receptor internalization. Moreover, in dynamic mass redistribution (DMR) assays that provide a pathway-agnostic readout of cellular responses, small molecule agonists produced robust responses. In contrast, Ang (1–7) failed to induce or block signaling in any of these assay platforms. We detected specific binding of radiolabeled Ang (1–7) to rat aortic endothelial cell (RAEC) membranes, but not to recombinant MAS1. Biphasic, concentration-dependent biased signaling responses to Ang II were detected in RAEC. These phases were associated with vastly different DMR characteristics and this likely provides a molecular basis for previously observed concentration-dependent divergent physiological actions of Ang II. Both phases of Ang II signaling in RAECs were potently inhibited by Ang (1–7), providing a plausible molecular mechanism for Ang (1–7) as counter regulator of the Ang II- AT1 axis, responsible at least in part for Ang (1–7) physiological activities.

Strong and sustained activation of the anticipatory unfolded protein response induces necrotic cell death.

The endoplasmic reticulum stress sensor, the unfolded protein response (UPR), regulates intracellular protein homeostasis. While transient activation of the reactive UPR by unfolded protein is protective, prolonged and sustained activation of the reactive UPR triggers CHOP-mediated apoptosis. In the recently characterized, evolutionarily conserved anticipatory UPR, mitogenic hormones and other effectors pre-activate the UPR; how strong and sustained activation of the anticipatory UPR induces cell death was unknown. To characterize this cell death pathway, we used BHPI, a small molecule that activates the anticipatory UPR through estrogen receptor α (ERα) and induces death of ERα+ cancer cells. We show that sustained activation of the anticipatory UPR by BHPI kills cells by inducing depletion of intracellular ATP, resulting in classical necrosis phenotypes, including plasma membrane disruption and leakage of intracellular contents. Unlike reactive UPR activation, BHPI-induced hyperactivation of the anticipatory UPR does not induce apoptosis or sustained autophagy. BHPI does not induce CHOP protein or PARP cleavage, and two pan-caspase inhibitors, or Bcl2 overexpression, have no effect on BHPI-induced cell death. Moreover, BHPI does not increase expression of autophagy markers, or work through recently identified programmed-necrosis pathways, such as necroptosis. Opening of endoplasmic reticulum IP3R calcium channels stimulates cell swelling, cPLA2 activation, and arachidonic acid release. Notably, cPLA2 activation requires ATP depletion. Importantly, blocking rapid cell swelling or production of arachidonic acid does not prevent necrotic cell death. Rapid cell death is upstream of PERK activation and protein synthesis inhibition, and results from strong and sustained activation of early steps in the anticipatory UPR. Supporting a central role for ATP depletion, reversing ATP depletion blocks rapid cell death, and the onset of necrotic cell death is correlated with ATP depletion. Necrotic cell death initiated by strong and sustained activation of the anticipatory UPR is a newly discovered role of the UPR.

Structural and Aggregation Properties of Alpha-Synuclein Linked to Phospholipase A2 Action.

Alpha-synuclein is a protein involved in the pathogenesis of Parkinson's disease. In vitro observations have shown that specific brain-enriched polyunsaturated fatty acids, such as arachidonic acid, can give rise to a conformational change in alpha-synuclein and ultimately induce its fibrillation. Arachidonic acid is released by phospholipase A2 activity and clinical observations have shown a link between mutations in PLA2G6, the gene responsible for the production of phospholipase A2, and early-onset types of parkinsonism. It is unknown how phospholipase A2-driven release of arachidonic acid can affect the conformation of alphasynuclein. The main objective of this study was to investigate if phospholipase A2-induced release of arachidonic acid can induce changes in conformation and aggregation state of alpha-synuclein. Recombinant human alpha-synuclein was expressed and isolated and incubated in the presence of phosphatidylcholine and phosphatidylserine (PC/PS) containing liposomes. The release of free fatty acids from PC/PS liposomes by bee venom phospholipase A2 was measured with the fluorescent probe acrylodated intestinal fatty acid-binding protein (ADIFAB) and radioactive labelling by preparing liposomes in the presence of L- 3-phosphatidylcholine, 1-stearyl-2[1-14C] arachidonoyl. The effect of free fatty acid release on the conformation of alpha-synuclein was assayed by far-UV circular dichroism and resistance against V8 protease-induced limited proteolysis. Aggregation of alpha-synuclein upon exposure to phospholipase A2-induced action on PC/PS liposomes was measured using thioflavin T fluorescence, SDS-PAGE, gel filtration chromatography, and transmission electron microscopy. RAW264.7 cells were transiently transfected with human alpha-synuclein and release of arachidonic acid was quantified using radiolabeling and liquid scintillation counting. Phospholipase A2 is capable of releasing arachidonic acid from biomimetic phospholipid membranes. Exposure of alpha-synuclein to phospholipase A2-induced release of arachidonic acid from PC/PS liposomes induces a conformational transition of the protein and leads to partial resistance against proteolytic cleavage by V8 protease. Prolonged incubation of alpha-synuclein with arachidonic acid, derived from PC/PS liposomes by phospholipase A2 leads to aggregate formation. In line with this, transiently transfected RAW264.7 cells with alpha-synuclein showed arachidonic acid release and punctate alpha-synuclein staining upon phospholipase A2 activation. The ability of arachidonic acid to drive alpha-synuclein to aggregate was independent of its oxidation state. We present data that suggest a biological context for the previously reported clinical observation that linked mutations in PLA2G6, the gene responsible for the production of phospholipase A2, and early-onset types of parkinsonism. Release of arachidonic acid, independent of its oxidation state, through activation of phospholipase A2-driven hydrolysis of phospholipid membranes, leads to the structural transition and aggregation of alpha-synuclein.

Computational Exploration of Natural Compounds to Target Cytosolic Phospholipase A 2 Protein: A Novel Therapeutic Target for Spinal Cord Injury.

Cytosolic Phospholipase A2 (cPLA2), an important isoform of PLA2 that mediates the release of arachidonic acid, plays a role in the pathogenesis of Spinal Cord Injury (SCI). The expression and activation of Cpla2 are significantly higher in SCI, leading to neuronal death in spinal cord tissue. Novel strategies are needed to substantially reverse the effect of cPLA2 activation; one such strategy is inhibiting cPLA2 by jamming its lipid binding C2 domain. To develop a much needed strategy to treat SCI, we used a Computer Aided Drug Design (CADD) method to discover novel cPLA2 inhibitors. we used a natural chemiome database for virtual screening, from which we selected the compounds exhibiting the greatest drug-likeliness properties for molecular docking simulation analysis. We studied the interaction of lead compounds at the atomic level; the results yielded a cPLA2 inhibitor of natural origin with the potential for ameliorating secondary tissue damage and promoting recovery of function after SCI. The top compound, lead 4exibited a binding energy of -10.02 Kcal/mol and formed three hydrogen bonds with the lipid binding C2 domain of the cPLA2 protein. An evaluation of cell cytotoxicity revealed an IC50 for lead4 of 134.2 ± 6.8 µM. An in-vitro analysis of lead4 is indicated anti-apoptotic activity via a decrease in caspase-3 expression. We used the CADD method to make a novel lead discovery for the treatment of SCI using compounds of natural origin. The selected natural compounds are non-toxic promising drugs against cPLA2 protein, allowing us to limits our focus on single compound for future in-vitro and invivo testing.

Epigenetic Regulation of the COX-2/PGE2 Pathway in Macrophages Results in Impaired Wound Healing in Diabetes

Abstract Macrophage (Mϕ) plasticity, allowing for transition of Mϕs from an inflammatory to a reparative phenotype, is critical for normal wound healing. In pathologic conditions, such as type 2 diabetes (T2D), wounds fail to heal due to impaired resolution of inflammation. There is increasing evidence that epigenetic-based mechanisms, including DNA hypomethylation, control Mϕ function. The cyclooxygenase (COX)-2/Prostaglandin E2 (PGE2) axis as well as upstream pathways including cytosolic phospholipase (c(PL)A2), involved in the release arachidonic acid, have been associated with chronic inflammation, however they have not been examined in diabetic wounds. The purpose of this study was to investigate if epigenetic modifications alter gene expression of the COX-2/PGE2 and c(PL)A2 pathway leading to derangements in macrophage inflammation in diabetes. Using the murine diet induce obesity (DIO) model, we evaluated diabetic wound healing compared with control mice. We demonstrate that TGFβ is elevated in DIO wound Mϕs in comparison to control wound Mϕs. Further, the increase in TGFβ stimulates miR-29B production in wound Mϕs resulting in hypomethylation and overexpression of the COX-2 gene. This results in diabetic wound Mϕs displaying increased levels of COX-2 which shunts amino acid metabolism towards the COX-2 pathway increasing PGE2 while limiting leukotriene synthesis via 5-lipoxygenase (5-LO) pathways. Lastly, c(PL)A2 appears to be upregulated in diabetic wound Mϕs and human diabetic monocytes. In summary, these results demonstrate that the COX-2/PGE2 pathway is increased in diabetic wound Mϕs by elevated TGFβ/miR-29b and results in elevated PGE2 that drives Mϕ-mediated inflammation preventing wound repair.

Abstract 529: Digoxin and Platelet Activation in Atrial fibrillation Patients

Background: digoxin use was shown to be associated with an increased risk of cardiovascular events In atrial fibrillation (AF) . We hypothesized that digoxin may affect cardiovascular risk by increasing platelet activation. Methods: This is a post-hoc analysis from an observational prospective study of AF patients treated with vitamin K antagonists. Patients were divided into two groups balanced for age, sex and cardiovascular risk factors: digoxin users (n=132) and non-users (n=388). Urinary excretion of 11-dehydro-thromboxane B 2 (TxB 2 ), a marker of platelet activation and serum digoxin concentration (SDC) were measured. In vitro experiments were performed on platelets from healthy subjects treated with scalar doses of digoxin (0.6 - 2.4 ng/ml). Results: Mean age of digoxin users was 75.2±7.2 years and 47.7% were women. Median overall 11-dehydro-TxB 2 was 105.0 (IQR 60.0-190.0) ng/mg creatinine, and median SDC was 0.65 (IQR 0.40-1.00) ng/ml. Urinary 11-dehydro-TxB 2 and SDC were significantly correlated (rS=0.350, p&lt;0.001). Patients in the upper tertile of SDC showed significantly higher urinary11-dehydro-TxB 2 compared to non-digoxin users (p=0.019). In vitro study showed that digoxin at concentration ≥1.2 ng/ml induced calcium mobilization, arachidonic acid (AA) release, TxB 2 biosynthesis and platelet aggregation, that were inhibited by the calcium chelator Ethylenediaminetetraacetic acid (EDTA) or by the specific phospholipase A 2 (PLA 2 ) inhibitor Arachidonyl trifluoromethyl ketone (ACOOCF3). Conclusion: We found a significant in vivo correlation between SDC and platelet activation. In vitro study showed that supra-therapeutic digoxin concentrations increased platelet aggregation via calcium-related PLA 2 phosphorylation. This finding provides new insights into the association between digoxin use and cardiovascular complications in AF patients.

Platelet-Activating Factor (PAF) Receptor Antagonism Modulates Inflammatory Signaling in Experimental Uveitis

ABSTRACTBackground: The phospholipid mediator platelet-activating factor (PAF) activates an inflammatory response that includes arachidonic acid release and prostaglandin production in the eye, increasing vascular permeability and inflammation.The purpose of this study is to investigate the action of LAU-0901, a novel PAF receptor antagonist, on experimental uveitis.Methods: Uveitis was induced in Lewis rats by lipopolysaccharide treatment. LAU-0901 was then delivered systemically in different concentrations at plus 4 and 16 hours, or vehicle injected as controls. Additional animals were used for histological analyses of untreated, uveitis, and uveitis-plus-LAU-0901 retinas. Conventional histological and immunohistochemical methods were employed. A slit lamp and Spectral Domain-Ocular Coherence Tomography (SD-OCT) retinal imager was used for anterior segment photography and posterior pole OCT. Rats were euthanized 4 hours after the second LAU-0901 injection in this 24-hour model. Aqueous humor was collected and quantified, and also analyzed for tumor necrosis factor alpha (TNF-α).Results: Uveitic eyes demonstrated hypopyon formation, leukocyte infiltration, and an increase in aqueous protein and TNF-α levels. LAU-0901 treatment resulted in a dose-dependent reduction in inflammation, reflected by reduced total protein levels (up to a 64% reduction). Moreover, hypopyon was prevented, leukocytes were absent in vitreous and aqueous humor, and TNF-α levels were reduced by 91%.Conclusions: The PAF receptor antagonist LAU-0901 decreases ocular inflammation in a rat model of anterior uveitis in a dose-dependent manner, suggesting that use of this molecule may provide a means to attenuate inflammation onset and offer a future alternative or adjunctive treatment for ocular inflammation.

Novel crystal structure of calcium independent phospholipase iPLA2β: mechanism of activity regulation and membrane localization

Calcium-independent phospholipase iPLA2β (also known as PLA2G6 or PNPLA9) is a signaling enzyme which hydrolyzes phospholipids to generate potent lipid second messengers in response to stress or injury. The enzyme is a product of the PARK14 gene with strong genetic link to a spectrum of neurodegenerative disorders including Parkinson's disease (PD). Alterations in iPLA2β function have demonstrated its role in other human pathologies including cardiovascular disease, cancer and diabetes. Mechanisms of its activation and tissue-specific functions remain poorly understood. This contrasts with known enzymatic activity and several well-characterized signaling cascades implicated in agonist-induced arachidonic acid release, insulin secretion, vascular constriction/relaxation, store-operated calcium-entry, cellular proliferation, migration and autophagy. We have solved a crystal structure of the full-length mammalian iPLA2β (short variant). The crystal structure significantly revises existing mechanistic models. It demonstrated unexpected oligomeric structure and the conformation of catalytic and auxiliary protein-interaction domains. The structure suggests the mechanisms of inhibition by calmodulin, activation through the autoacylation reaction and the potential role of ATP in stabilizing ankyrin repeats. The novel crystal structure together with biochemical studies has immediate implications for the mechanisms of the phospholipase activity, of the inhibition and activation as well as of the potential mechanism of tissue specific cellular localization. It provides a well-defined framework to investigate the role of neurodegenerative mutations and the function of iPLA2β in the brain as well as its role in other diseases. Support or Funding Information American Heart Association Grant-in-Aid #0665513Z, Center for Advancement of Science in Space (CASIS) grant CASIS-2012-1, NIH/NINDS grant R21NS094854, to S.K. The mechanism of iPLA2b inhibition by calmodulin (a) and activation and interaction with membrane and membrane proteins (b). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mustard analogs of acetylcholine: irreversible partial agonists at Gq‐linked muscarinic receptors

Mustard analogs of many receptor ligands have been used to irreversibly label or inactivate receptors. In aqueous solution, mustard groups cyclize to form aziridinium ions that closely mimic quaternary amines. Such a reactive intermediate may therefore bind in the same orientation within the same binding pocket of the receptor that accommodates the related reversible ligand. Once oriented, it is capable of alkylating any of a number of nearby amino acid side chains (including those of cysteine, glutamate, aspartate, and others) and might, in principle, induce irreversible activation of the receptor. However, acetylcholine mustard has been reported by several laboratories to possess agonist activity at muscarinic receptors only until alkylation is achieved, at which point the receptor becomes inactivated. Therefore, we were surprised to find that agonist activity persisted after irreversible binding of acetylcholine mustard and its analog, acetylethylcholine mustard, at Gq‐linked muscarinic receptors (M1, M3, and M5) expressed in CHO cells. The cells were preincubated with cyclized agonist mustards and thoroughly washed prior to assay; the observed responses could be prevented by including atropine in the preincubation phase, but were not inhibited by the presence of atropine in the assay phase. The metabolism of inositol phosphates (IP) was enhanced robustly, though to a lesser degree than that induced by acetylcholine. Acetylethylcholine mustard was less efficacious than acetylcholine mustard, especially at the M5 subtype. For both mustards, the order of efficacy was M1&gt;M3&gt;M5. The most interesting results were obtained measuring the receptor‐stimulated release of labeled arachidonic acid (AA) from intact cells. After preincubation as above, the basal AA release was found to be elevated to a small but significant degree; we have previously shown amiodarone to be a positive allosteric modulator (PAM) at these receptors and inclusion of amiodarone in the assay phase dramatically increased the response of cells that had been preincubated with the agonist mustards. Once again, atropine in the assay itself did not prevent this effect of amiodarone, but atropine in the preincubation phase prevented it completely. At the M1 subtype, the well‐known M1‐specific PAM BQCA enhanced the mustard‐induced activity in a manner similar to that observed for amiodarone. These studies confirm that amiodarone, BQCA, and other ligands investigated act at an allosteric site and suggest that receptors that have been alkylated by acetylcholine mustard and acetylethylcholine mustard do adopt active conformations that can be further enhanced by appropriate allosteric ligands.Support or Funding InformationSupported AG005214 to JEThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glucocorticoids impair platelet thromboxane biosynthesis in community-acquired pneumonia

Previous reports suggest that community-acquired pneumonia (CAP) is associated with an enhanced risk of myocardial infarction (MI) and that enhanced platelet activation may play a role.Aims of this study were to investigate if urinary excretion of 11-dehydro-thromboxane (Tx) B2, a reliable marker of platelet activation in vivo, was elevated in CAP and whether glucocorticoid administration reduced platelet activation.Three-hundred patients hospitalized for CAP were recruited and followed-up until discharge. Within the first 2 days from admission, urinary 11-dehydro-TxB2 and serum levels of methylprednisolone and betamethasone were measured. 11-Dehydro-TxB2 was also measured in a control group of 150 outpatients, matched for age, sex, and comorbidities. Finally, in-vitro studies were performed to assess if glucocorticoids affected platelet activation, at the same range of concentration found in the peripheral circulation of CAP patients treated with glucocorticoids.Compared to controls, CAP patients showed significantly higher levels of 11-dehydro-TxB2 (110 [69–151] vs. 163 [130–225] pg/mg creatinine; p < 0.001). During the in-hospital stay, 31 patients experienced MI (10%). A COX regression analysis showed that 11-dehydro-TxB2 independently predicted MI (p = .005). CAP patients treated with glucocorticoids showed significantly lower levels of 11-dehydro-TxB2 compared to untreated ones (147 [120–201] vs. 176 [143–250] pg/mg creatinine; p < 0.001).In vitro, glucocorticoids-treated platelets showed a dose-dependent decrease of ADP-induced platelet aggregation, TxB2 production, cPLA2 phosphorylation and arachidonic acid release from the platelet membrane.In conclusion, platelet TxB2 is overproduced in CAP patients and may be implicated in MI occurrence. Glucocorticoids reduce platelet release of TxB2 in vitro and urinary excretion of 11-dehydro-TxB2 in vivo and may be a novel tool to decrease platelet activation in this setting.

In vitro profiling of toxic effects of prominent environmental lower-chlorinated PCB congeners linked with endocrine disruption and tumor promotion

The mechanisms contributing to toxic effects of airborne lower-chlorinated PCB congeners (LC-PCBs) remain poorly characterized. We evaluated in vitro toxicities of environmental LC-PCBs found in both indoor and outdoor air (PCB 4, 8, 11, 18, 28 and 31), and selected hydroxylated metabolites of PCB 8, 11 and 18, using reporter gene assays, as well as other functional cellular bioassays. We focused on processes linked with endocrine disruption, tumor promotion and/or regulation of transcription factors controlling metabolism of both endogenous compounds and xenobiotics. The tested LC-PCBs were found to be mostly efficient anti-androgenic (within nanomolar – micromolar range) and estrogenic (at micromolar concentrations) compounds, as well as inhibitors of gap junctional intercellular communication (GJIC) at micromolar concentrations. PCB 8, 28 and 31 were found to partially inhibit the aryl hydrocarbon receptor (AhR)-mediated activity. The tested LC-PCBs were also partial constitutive androstane receptor (CAR) and pregnane X receptor (PXR) agonists, with PCB 4, 8 and 18 being the most active compounds. They were inactive towards other nuclear receptors, such as vitamin D receptor, thyroid receptor α, glucocorticoid receptor or peroxisome proliferator-activated receptor γ. We found that only PCB 8 contributed to generation of oxidative stress, while all tested LC-PCBs induced arachidonic acid release (albeit without further modulations of arachidonic acid metabolism) in human lung epithelial cells. Importantly, estrogenic effects of hydroxylated (OH-PCB) metabolites of LC-PCBs (4-OH-PCB 8, 4-OH-PCB 11 and 4′-OH-PCB 18) were higher than those of the parent PCBs, while their other toxic effects were only slightly altered or suppressed. This suggested that metabolism may alter toxicity profiles of LC-PCBs in a receptor-specific manner. In summary, anti-androgenic and estrogenic activities, acute inhibition of GJIC and suppression of the AhR-mediated activity were found to be the most relevant modes of action of airborne LC-PCBs, although they partially affected also additional cellular targets.

Protection of bovine mammary epithelial cells from hydrogen peroxide-induced oxidative cell damage by selenium

The uncontrolled release of arachidonic acid (ARA) and its metabolism by lipoxygenase (LOX) pathway can induce and aggravate cellular oxidative stress. Selenium (Se) is an integral part of some antioxidative selenoproteins and may protect cells from oxidative damage by modulating ARA release and metabolism. The present study aimed to investigate the protective response of Se against hydrogen peroxide (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)-induced oxidative damage in bovine mammary epithelial cells (BMECs). The BMECs were incubated for 24 h in serum-free medium and then divided into four groups randomly. The cells in groups 1 and 2 were subsequently incubated for 30 h in serum-free medium containing 0 (control) and 50 nM Se (Se treatment group). The cells in groups 3 and 4 were incubated for 24 h in serum-free medium containing 0 and 50 nM Se, and then treated with 600 μM H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; for 6 h (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; damage group and Se prevention group). The results showed that Se attenuated the H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;-induced production of reactive oxygen species and the decrease of antioxidative enzymes as glutathione peroxidase (GPX), thioredoxin reductase (TrxR), selenoprotein P (SelP), superoxide dismutase, and catalase in BMECs. The preventive effects of Se on the decrease of selenoprotein activity were demonstrated further by the increase of mRNA expression for GPX1, TrxR1, and SelP, and protein expression for GPX1 and TrxR1. Pretreatment of cells with Se inhibited the H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;-induced increase of mRNA expressions and activities for cytosolic phospholipase A2 and 5-lipoxygenase, ARA release, and 15-hydroperoxyeicosatetraenoic acid production. Se also blocked the H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;-induced activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase but not that of extracellular signal-regulated kinase. These results suggested that Se may protect BMECs against H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;-induced oxidative damage by increasing selenoproteins synthesis, inhibiting MAPK pathway, and then decreasing ARA release and its metabolism by LOX pathway.