Cytosolic Phospholipase A2 Inhibitor Research Articles

Mechanism of glutamine inhibition of cytosolic phospholipase a2 (cPLA2 ): Evidence of physical interaction between glutamine-Induced mitogen-activated protein kinase phosphatase-1 and cPLA2.

Non-essential amino acid L-glutamine (Gln) possesses anti-inflammatory activity via deactivating cytosolic phospholipase A2 (cPLA2 ). We showed previously that Gln deactivated cPLA2 indirectly via dephosphorylating p38 mitogen-activated protein kinase (MAPK), the major kinase for cPLA2 phosphorylation, through inducing MAPK phosphatase-1 (MKP-1). In this study, we investigated the precise mechanism underlying Gln deactivation of cPLA2 . In lipopolysaccharide (LPS)-treated mice, Gln injection resulted in dephosphorylation of phosphorylated cPLA2 (p-cPLA2 ), which coincided with rapid Gln induction of MKP-1. MKP-1 small interfering RNA (siRNA) abrogated the ability of Gln to induce MKP-1 as well as the dephosphorylation of cPLA2 . Co-immunoprecipitation and in-situ proximity ligation assay revealed a physical interaction between MKP-1 and p-cPLA2 . In a murine model of allergic asthma, we also demonstrated the physical interaction between MKP-1 and p-cPLA2 . Furthermore, Gln suppressed various allergic asthma phenotypes, such as neutrophil and eosinophil recruitments into the airway, airway levels of T helper type 2 (Th2) cytokines [interleukin (IL)-4, IL-5 and IL-13], airway hyperresponsiveness, mucin production and metabolites (leukotriene B4 and platelet-activating factor) through inhibiting cPLA2 in a MKP-1-dependent manner. These data suggest that MKP-1 uses cPLA2 , in addition to p38, as a substrate, which further potentiates the anti-inflammatory action of Gln.

Involvement of microsomal NADPH-cytochrome P450 reductase in metabolic reduction of drug ketones.

Recently, it was found that the carbonyl group of 1-[3-(4-phenoxyphenoxy)-2-oxopropyl]indole-5-carboxylic acid (5), an inhibitor of the pro-inflammatory enzyme cytosolic phospholipase A2 α, is easily reduced by rat liver S9 fractions in vitro. Determination of the inhibitory potency of certain putative inhibitors of carbonyl reducing enzymes on the transformation of the ketone derivative 5 to its alcohol 6 by recombinant microsomal NADPH-cytochrome P450 reductase and by recombinant cytosolic carbonyl reductase-1 now reveals that these compounds show a lack of specificity for these two enzymes in part. Thus, an assignment of the roles of different carbonyl reductases in metabolic keto reduction by the use of inhibitors is problematic. In addition, the ability of NADPH-cytochrome P450 reductase and carbonyl reductase-1 to reduce the ketone groups of the drugs haloperidol and daunorubicin was examined. Under the conditions applied, a pronounced reductive metabolism was only observed for daunorubicin in the presence of microsomal NADPH-cytochrome P450 reductase. Similarly, in rat liver S9 fractions a marked reduction of daunorubicin was seen, while haloperidol was only slightly metabolized to its alcohol. After separation of the S9 homogenate into a microsomal and a cytosolic fraction, it became evident that the ketone groups of daunorubicin, haloperidol and compound 5 were mainly reduced by cytosolic enzymes. However, since microsomes also catalysed these carbonyl reductions to some extent, it can be concluded that microsomal NADPH-cytochrome P450 reductase can contribute to metabolic keto reductions in xenobiotics. Copyright © 2015 John Wiley & Sons, Ltd.

Evaluation of drug–drug interaction between the novel cPLA2 inhibitor AK106-001616 and methotrexate in rheumatoid arthritis patients

1. Drug interaction potential between AK106-001616, a novel cytosolic phospholipase A2 inhibitor, and methotrexate (MTX) in rheumatoid arthritis patients was investigated. This trial is registered with ClinicalTrials.gov, number NCT00902369.2. In the clinical study, the 90% confidence intervals (CIs) for the geometric mean ratio (GMR) of AUC0–t of MTX administered after AK106-001616 200 mg compared to the MTX without AK106-001616 were within 80–125%. However, administration of AK106-001616 at doses of 400 and 600 mg exceeded the 125% threshold. As small but statistically significant increases in AUC0–t were observed, we investigated the mechanism for this drug–drug interaction between MTX and AK106-001616.3. In vitro, AK106-001616 inhibited OAT1 (IC50 = 18.4 μM, Ki = 33.6 μM) in a non-competitive manner and OAT3 (IC50 = 1.80 μM, Ki = 1.49 μM) in a competitive manner. Both transporters are involved in MTX transport in renal proximal tubules.4. AK106-001616 has a weak drug interaction with MTX. In vitro studies provide a mechanistic understanding of the in vivo inhibition of transporters by AK106-001616.

Bromoenol Lactone, an Inhibitor of Calcium-Independent Phospholipase A2, Suppresses Carrageenan-Induced Prostaglandin Production and Hyperalgesia in Rat Hind Paw.

Prostaglandin (PG) E2 and PGI2 are essential to hyperalgesia in inflammatory tissues. These prostaglandins are produced from arachidonic acid, which is cleaved from membrane phospholipids by the action of phospholipase A2 (PLA2). Which isozyme of PLA2 is responsible for the cleavage of arachidonic acid and the production of prostaglandins essential to inflammation-induced hyperalgesia is not clear. In this study, we examined the effects of two PLA2 isozyme-specific inhibitors on carrageenan-induced production of PGE2 and PGI2 in rat hind paw and behavioral nociceptive response to radiant heat. Local administration of bromoenol lactone (BEL), an inhibitor of calcium-independent PLA2 (iPLA2), significantly reduced carrageenan-induced elevation of prostaglandins in the inflamed foot pad 3 h after injection. It also ameliorated the hyperalgesic response between 1 h and 3 h after carrageenan injection. On the other hand, AACOCF3, an inhibitor of cytosolic PLA2, suppressed neither prostaglandin production nor the hyperalgesic response. BEL did not suppress the mRNA levels of iPLA2 β, iPLA2 γ, cyclooxygenase-2, microsomal prostaglandin E synthase, prostaglandin I synthase, or proinflammatory cytokines in the inflamed foot pad, indicating that BEL did not suppress inflammation itself. These results suggest that iPLA2 is involved in the production of prostaglandins and hyperalgesia at the inflammatory loci.

FTY720 impairs CD8 T-cell function independently of the sphingosine-1-phosphate pathway

Fingolimod (FTY720) is a multiple sclerosis (MS) therapeutic that upon phosphorylation causes the internalization of sphingosine-1-phosphate receptors (S1PR) and traps CCR7+ T-cells in lymph nodes but relatively spares CCR7-effector T-cells. Nonetheless, FTY720-treated patients are more susceptible to viral infections, indicating a CD8 T-cell defect. Thus, the effects of FTY720 on CD8 T-cells were investigated. To this end, we utilized experimental autoimmune encephalomyelitis (EAE) and a murine influenza model. CD8 T-cell trafficking, IFNγ and Granzyme B (GrB) production were assessed by flow cytometry. CD8 T-cell cytotoxic function was assessed in vitro by an LDH release assay. FTY720 not only ameliorated EAE by sequestering T-cells, but also reduced IFNγ and Granzyme B (GrB) in splenic CD8 T-cells. Murine influenza infection was exacerbated and mortality was increased, as FTY720 inhibited CD8 T-cell GrB production and lung infiltration. Remarkably, only the unphosphorylated compound was able to reduce IFNγ and GrB levels in CD8 T-cells and inhibits their cytotoxic function in vitro. The phosphorylated moiety had no effect in vitro, indicating that CD8 T-cell suppression by FTY720 is independent of S1PR modulation. The addition of arachidonic acid rescued CD8 T-cell function, suggesting that this effect may be mediated via inhibition of cytosolic phospholipase A2. Herein, we demonstrate that FTY720 suppresses CD8 T-cells independently of its trafficking effects and S1PR modulation. This provides a novel explanation not only for the increased rate of viral infections in FTY720-treated patients, but also for its efficacy in MS, as CD8 T-cells have emerged as crucial mediators of MS pathogenesis.

Protective effect of cytosolic phospholipase A2 inhibition against inflammation and degeneration by promoting regulatory T cells in rats with experimental autoimmune encephalomyelitis.

Cytosolic phospholipase A2 (cPLA2) is the rate-limiting enzyme that initiates the production of various inflammatory mediators. Previous studies have shown that inhibiting cPLA2 exerts a neuroprotective effect on experimental autoimmune encephalomyelitis (EAE) by ameliorating the severity of the disease and influencing Th1 and Th17 responses. However, it remains unclear whether treatment with a cPLA2 inhibitor will influence the regulatory T cells (Tregs) that play a critical role in maintaining immune homeostasis and preventing autoimmune diseases. In this study, the cPLA2 inhibitor AX059 reduced the onset and progression of EAE in Lewis rats. In addition, this effect was accompanied by activation of Tregs and alterations in the expression of their various cytokines. The study therefore demonstrated that Tregs are involved in the immunomodulatory effect mediated by cPLA2 inhibition. These findings may have clinical application in the treatment of multiple sclerosis.

Anti-proliferative efficacy comparison of conjugated linoleic acid on human cancer cell lines

The anti-proliferative efficacy of t,t-conjugated linoleic acid (t,t-CLA), c9,t11-CLA, and t10,c12-CLA was compared in several human cancer cell lines. Gastric NCI-N87, liver Hep3B, pancreas Capan-2, and lung NCI-H522 cancer cells were incubated with 50 μM CLA isomers over a period of 6 days. The t,t-CLA inhibited the growth of all cancer cell lines to different extents, but c9,t11-CLA and t10,c12-CLA inhibited or stimulated the growth of the cancer cell lines. NCI-N87 cells were the most sensitive to growth inhibition and apoptosis from all CLA isomers tested. In NCI-N87 cells, CLA isomers reduced the release of arachidonic acid (AA) via the inhibition of cytosolic phospholipase A2 (cPLA2 ) activity, consequently reducing the production of PGE2 through the inhibition of cyclooxygenase-2 (COX-2). The efficacies of CLA isomers were in the following order (from most to least effective): t,t-CLA, t10,c12-CLA and c9,t11-CLA. Overall, these results imply that the anti-proliferative efficacy of t,t-CLA on cancer cells, especially NCI-N87 cells, was greater than other CLA isomers due to its induction of apoptosis through the inhibition of cPLA2 and COX-2 activities.

Inhibitors of arachidonate-regulated calcium channel signaling suppress triggered activity induced by the late sodium current

Disturbances in myocyte calcium homeostasis are hypothesized to be one cause for cardiac arrhythmia. The full development of this hypothesis requires (i) the identification of all sources of arrhythmogenic calcium and (ii) an understanding of the mechanism(s) through which calcium initiates arrhythmia. To these ends we superfused rat left atria with the late sodium current activator type II Anemonia sulcata toxin (ATXII). This toxin prolonged atrial action potentials, induced early afterdepolarization, and provoked triggered activity. The calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 (N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphon-amide) suppressed ATXII triggered activity but its inactive congener KN-92 (2-[N-(4-methoxy benzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) did not. Neither drug affected normal atrial contractility. Calcium entry via L-type channels or calcium leakage from sarcoplasmic reticulum stores are not critical for this type of ectopy as neither verapamil ((RS)-2-(3,4-dimethoxyphenyl)-5-{[2-(3,4-dimethoxyphenyl)ethyl]-(methyl)amino}-2-prop-2-ylpentanenitrile) nor ryanodine affected ATXII triggered activity. By contrast, inhibitors of the voltage independent arachidonate-regulated calcium (ARC) channel and the store-operated calcium channel specifically suppressed ATXII triggered activity without normalizing action potentials or affecting atrial contractility. Inhibitors of cytosolic calcium-dependent phospholipase A2 also suppressed triggered activity suggesting that this lipase, which generates free arachidonate, plays a key role in ATXII ectopy. Thus, increased left atrial late sodium current appears to activate atrial Orai-linked ARC and store operated calcium channels, and these voltage-independent channels may be unexpected sources for the arrhythmogenic calcium that underlies triggered activity.

Abstract P6-04-08: Cytosolic phospholipase A2 (cPLA2) as a therapeutic target in basal-like breast cancer

Abstract Introduction: Basal-like breast cancer is frequently associated with triple negative phenotype, and there is a need for novel therapeutic strategies for this patient population. Inhibitors of cytosolic phospholipase A2 (cPLA2) have been suggested to block both MAPK and PI3K signalling and have a high potential for activity in basal-like breast cancer (Lin 1993, Wen 2013). In this study, we compared the expression of PLA2G4A between luminal B and basal-like breast cancer, both in patient-derived xenograft models (PDX) and human cancer tissue. In addition, we studied the effect of the novel cPLA2 inhibitor AVX235 on tumor growth in a basal-like PDX model. Materials and methods: Tumor tissue specimens were obtained from PDX models (n = 26) and a clinical breast cancer biobank (n = 32). Gene expression analysis was carried out on Agilent 8×60K microarrays. The expression of 54 genes directly involved in choline metabolism was examined. Differential expression of choline genes between basal-like and luminal B tumors was calculated by subtraction of log2 expression values. Mice carrying bilateral MAS98.12 basal-like xenografts (Bergamaschi 2009) were treated with the cPLA2 inhibitor AVX235 (30 mg/kg i.p. daily for 7 days then every second day for 14 days, n = 6) or drug-free vehicle (control group, n = 6). Results: The PDX models were subtyped into basal-like (n = 19) and luminal B (n = 7) subtypes based on the expression of 500 intrinsic genes [Sørlie 2003]. For the human cancer tissue, there were 18 basal-like and 14 luminal B tumors. There was a significant correlation between differential choline gene expression in basal-like vs luminal B tumors in PDX models and human cancer tissue (p<1.3*10−12). Both in PDX models (p<0.04) and human cancer tissue (p<0.0003), PLA2G4A was significantly higher expressed in basal-like than luminal B tumors. Treatment with AVX235 markedly reduced the growth rate of MAS98.12 xenografts compared to controls. After 19 treatmment days, the mean tumor volume (normalised to volume at start of treatment) in the treatment group was 36% of the tumor volume in the control group, the difference being statistically significant (p = 0.023). No signs of treatment-related adverse effects were observed. Conclusion: PLA2G4A is higher expressed in basal-like than in luminal B breast cancer. Treatment with the cPLA2 inhibitor AVX235 significantly inhibits tumor growth. These data suggest that cPLA2 inhibitors may be of particular value in treatment of basal-like breast cancer.

Bradykinin Contracts Rat Urinary Bladder Largely Independently of Phospholipase C

Several receptor systems in the bladder causing detrusor smooth muscle contraction stimulate phospholipase C (PLC). PLC inhibition abolishes bladder contraction via P2Y6 but not that via M3 muscarinic receptors, indicating a receptor-dependent role of PLC. Therefore, we explored the role of PLC in rat bladder contraction by bradykinin. The PLC inhibitor U 73,122 [1-(6-[([17β]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-1H-pyrrole-2,5-dione] did not affect the bradykinin response to a significantly greater degree than its inactive analog U 73,343 [10 μM each; 1-(6-[-([17β]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-2,5-pyrrolidinedione], whereas the phospholipase D inhibitor butan-1-ol relative to its inactive control butan-2-ol caused a weak but significant inhibition (0.3% each). The cytosolic phospholipase A2 inhibitor arachidonyltrifluoromethyl ketone (300 μM) and the cyclooxygenase inhibitor indomethacin (10 μM) caused strong inhibition of the bradykinin response. The L-type Ca(2+) channel blocker nifedipine (10-100 nM) concentration-dependently caused strong inhibition, whereas only a small but significant inhibition was seen with SK&F 96,365 [10 μM; 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl], an inhibitor of receptor-operated Ca(2+) channels. Several protein kinase C inhibitors yielded an equivocal picture (inhibition by 10 μM bisindolylmaleimide I and 1 μM calphostin but not by 10 μM chelerythrine). The rho kinase inhibitor Y 27,632 [1-10 μM; trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide] caused a strong and concentration-dependent inhibition of the bradykinin response. Our data support that not only M3 but also bradykinin receptors cause bladder contraction by a largely PLC-independent mechanism. Both responses strongly involve L-type Ca(2+) channels and rho kinase, whereas only the bradykinin response additionally involves the phospholipase A2/cyclooxygenase pathway.

Cytosolic PhospholipaseA2 Inhibition with PLA-695 Radiosensitizes Tumors in Lung Cancer Animal Models

Lung cancer remains the leading cause of cancer deaths in the United States and the rest of the world. The advent of molecularly directed therapies holds promise for improvement in therapeutic efficacy. Cytosolic phospholipase A2 (cPLA2) is associated with tumor progression and radioresistance in mouse tumor models. Utilizing the cPLA2 specific inhibitor PLA-695, we determined if cPLA2 inhibition radiosensitizes non small cell lung cancer (NSCLC) cells and tumors. Treatment with PLA-695 attenuated radiation induced increases of phospho-ERK and phospho-Akt in endothelial cells. NSCLC cells (LLC and A549) co-cultured with endothelial cells (bEND3 and HUVEC) and pre-treated with PLA-695 showed radiosensitization. PLA-695 in combination with irradiation (IR) significantly reduced migration and proliferation in endothelial cells (HUVEC & bEND3) and induced cell death and attenuated invasion by tumor cells (LLC &A549). In a heterotopic tumor model, the combination of PLA-695 and radiation delayed growth in both LLC and A549 tumors. LLC and A549 tumors treated with a combination of PLA-695 and radiation displayed reduced tumor vasculature. In a dorsal skin fold model of LLC tumors, inhibition of cPLA2 in combination with radiation led to enhanced destruction of tumor blood vessels. The anti-angiogenic effects of PLA-695 and its enhancement of the efficacy of radiotherapy in mouse models of NSCLC suggest that clinical trials for its capacity to improve radiotherapy outcomes are warranted.

THU0223 The Novel CPLA2 Inhibitor AK106-001616 is a New Category of Anti-Inflammatory/Analgesic Drug Demonstrating Efficacy and Favorable Tolerability in the Treatment of Rheumatoid Arthritis

BackgroundAK106-001616 (AK106) is a selective cytosolic phospholipase A2 (cPLA2) inhibitor with a novel mechanism of action as an anti-inflammatory/analgesic drug. Unlike non-steroidal anti-inflammatory drugs (NSAIDs), AK106 inhibits the production of...

Dissecting fingolimod’s action: a novel immunomodulatory effect of the unphosphorylated compound on cytotoxic T cell function (P5159)

Abstract Fingolimod (FTY), a sphingosine-1-phosphate receptor modulator, is a multiple sclerosis (MS) therapeutic that upon phosphorylation traps CCR7+ T cells in lymph nodes (LN). CCR7- effector T cells are spared, thus allowing effective infection clearance. Nonetheless, FTY-treated patients are more susceptible to viral infections, indicating a cytotoxic T (Tc) cell defect. Yet, FTY’s effect on Tc cells remains unclear. To address this question, we utilized experimental autoimmune encephalomyelitis (EAE) and a murine influenza model. FTY ameliorated EAE by sequestering T cells, but also inhibited splenic Tc cell IFNγ and Granzyme B(GrB) expression. Influenza was exacerbated and mortality was increased, as FTY inhibited Tc cell activation and lung infiltration. To elucidate its mechanism of action, murine splenocyte cultures were treated with phosphorylated and unphosphorylated FTY, as both exist in vivo. Remarkably, only the latter reduced IFNγ and GrB levels in Tc cells and inhibited their function in an LDH cytotoxicity assay. The addition of arachidonic acid rescued Tc cell function, suggesting that FTY’s effect is due to inhibition of cytosolic phospholipase A2. Herein, we demonstrate that FTY suppresses Tc cell function independently of its cell trafficking effects. This provides a novel explanation not only for the increased rate of viral infections in FTY-treated patients, but also for its efficacy in MS, as Tc cells have emerged as crucial mediators of MS pathogenesis.

Regulation of connexin36 gap junction channels by n‐alkanols and arachidonic acid

We examined junctional conductance (gj) and its dependence on transjunctional voltage in gap junction (GJ) channels formed of wild-type connexin36 (Cx36) or its fusion form with green fluorescent protein (Cx36-EGFP) transfected in HeLa cells or endogenously expressed in primary culture of pancreatic β-cells. Only a very small fraction (∼0.8%) of Cx36-EGFP channels assembled into junctional plaques of GJs were open under control conditions. We found that short carbon chain n-alkanols (SCCAs) increased gj, while long carbon chain n-alkanols resulted in full uncoupling; cutoff is between heptanol and octanol. The fraction of functional channels and gj increased several fold under an exposure to SCCAs, or during reduction of endogenous levels of arachidonic acid (AA) by exposure to fatty acid-free BSA or cytosolic phospholipase A2 inhibitors. Moreover, uncoupling caused by exogenously applied AA can be rescued by BSA, which binds AA and other polyunsaturated fatty acids (PUFAs), but not by BSA modified with 1,2-cyclohexanedione, which does not bind AA and other PUFAs. We propose that under control conditions, Cx36 GJ channels in HeLa transfectants and β-cells are inhibited by endogenous AA, which stabilizes a closed conformational state of the channel that leads to extremely low fraction of functional channels. In addition, SCCAs increase gj by interfering with endogenous AA-dependent inhibition, increasing open probability and the fraction of functional channels.

A catalytically-inactive snake venom Lys49 phospholipase A2 homolog induces expression of cyclooxygenase-2 and production of prostaglandins through selected signaling pathways in macrophages

The effects of a snake venom Lys-49 phospholipase A2 (PLA2) homolog named MT-II, devoid of enzymatic activity, on the biosynthesis of prostaglandins and protein expression of cyclooxygenase-2 (COX-2) and signaling pathways involved were evaluated in mouse macrophages in culture and in peritoneal cells ex vivo. Stimulation of macrophages with MT-II leads to production of prostaglandin D2 (PGD2) and prostaglandin E2 (PGE2) and protein expression of COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1). Inhibition of cytosolic PLA2 (cPLA2), but not Ca2+ independent PLA2 (iPLA2) reduced release of PGD2 and PGE2 and expression of COX-2 induced by MT-II. Inhibition of nuclear factor κB (NF-κB) significantly reduced MT-II-induced PGE2, but not PGD2 production and COX-2 expression. Inhibitors of either protein kinase C (PKC), protein tyrosine kinase (PTK), or extracellular signal-regulated kinase (ERK) pathways abrogated MT-II-induced NF-κB activation and reduced COX-2 expression and PGE2 release, whereas the p38 mitogen-activated protein kinase (MAPK) inhibitor reduced MT-II-induced COX-2 expression and PGD2 production. Inhibition of phosphatidylinositol-3-kinase (PI3K) pathway abrogated MT-II-induced NF-κB activation, but affected neither prostaglandins production nor COX-2 expression. MT-II-induced production of PGD2 and PGE2 and COX-2 expression were also observed in vivo after intraperitoneal injection into mice. Collectively, our data demonstrate that a catalytically-inactive PLA2 homolog is capable of inducing prostaglandins biosynthesis and COX-2 expression in macrophages in both in vitro and in vivo models, indicating that the enzymatic activity of PLA2 is not necessary to trigger these effects. MT-II-activated NF-κB, cPLA2 and distinct protein kinases are the principal steps involved in these cellular events.

Sex-specific response of rat costochondral cartilage growth plate chondrocytes to 17β-estradiol involves differential regulation of plasma membrane associated estrogen receptors

Both male and female rat growth plate chondrocytes express estrogen receptors (ERs); however 17β-estradiol (E2) induces membrane responses leading to activation of phospholipase A2 (PLA2), phospholipase C (PLC), prostaglandin E2 (PGE2) production, protein kinase C (PKC), and ultimately mitogen protein kinase (MAPK) only in female cells. This study investigated if these sex-specific responses are due to differences in the actual ERs or in downstream signaling. Western blots and flow cytometry of costochondral cartilage resting zone chondrocytes (RCs) showed 2–3 times more ERα in plasma membranes (PMs) from female cells than male cells. Tunicamycin blocked E2-dependent ER-translocation to the PM, indicating palmitoylation was required. Co-immunoprecipitation showed E2 induced complex formation between ER isoforms only in female RCs. To examine if the lack of response in PKC and PGE2 in males is due to differences in signaling, we examined involvement of ERs and the role of PLC and PLA2. Selective ERα (propylpyrazole triol, PPT) and ERβ (diarylproprionitrile, DPN) agonists activated PKC in female RCs only. The PLC inhibitor, U73122 blocked E2's effect on PKC and the cytosolic PLA2 inhibitor, AACOCF3 inhibited the effect on PGE2 in female RCs, confirming involvement of PLC and PLA2 in the mechanism. The PLC activator, m-3M3FβS activated PKC and PLAA peptide increased PGE2 levels in male and female RCs, showing that the signaling pathways are present. These data indicate that differences in membrane ER amount, localization, translocation and interaction are responsible for the sexual dimorphic response to E2.

C2-di-ethyl-ceramide-1-phosphate as an inhibitor of group IVA cytosolic phospholipase A2

Ceramide-1-phosphate (C1P) has been shown to bind with C2 domain in group IVA cytosolic phospholipase A2 (cPLA2α, PLA2G4A) and activate the enzyme activity directly. In cells, C1P causes translocation of cPLA2α to perinuclear regions including the Golgi complex by interacting with C2 domain in the enzyme, and then cPLA2α releases arachidonic acid from substrate phospholipids in the regions. In this study, we synthesized new di-ethyl (DE) phosphate ester analogs of C1P with N-acyl chains of different lengths, and examined their effects on cPLA2α. A DE-C1P analog with a C2-N-acyl chain (C2-DE-C1P), but not DE-C1P analogs with longer N-acyl chain, such as C6- and C16-DE-C1P, inhibited release of arachidonic acid via cPLA2α activation in CHO-W11A cells expressing platelet-activating factor (PAF) receptors without changing secretory phospholipase A2-induced release. Treatment with C2-DE-C1P did not modify phosphorylation of extracellular signal-regulated kinase 1/2 and cPLA2α and increase of intracellular Ca2+ level induced by PAF, but inhibited Ca2+- and PAF-induced accumulation of cPLA2α in the Golgi complex. Phosphatidylcholine vesicles containing C2-DE-C1P reduced cPLA2α activity in vitro. C2-DE-C1P disturbed the binding of the enzyme to glycerophospholipids in the lipid–protein overlay assay, and the reagent alone did not bind to the enzyme. Interestingly, C2-DE-C1P inhibited neither Ca2+- and PAF-induced accumulation of C2 domain of cPLA2α in the Golgi complex nor binding of cPLA2α to C16-C1P. These results suggest that C2-DE-C1P appeared to inhibit cPLA2α, probably by interaction with a site in the catalytic domain of the enzyme, not with the site in C2 domain responsible for native C1P.

Activity-Induced Long-Term Potentiation of Excitatory Synapses in Developing Zebrafish Retina In Vivo

Neural activity-induced long-term potentiation (LTP) of synaptic transmission is believed to be one of the cellular mechanisms underlying experience-dependent developmental refinement of neural circuits. Although it is well established that visual experience and neural activity are critical for the refinement of retinal circuits, whether and how LTP occurs in the retina remain unknown. Using in vivo perforated whole-cell recording and two-photon calcium imaging, we find that both repeated electrical and visual stimulations can induce LTP at excitatory synapses formed by bipolar cells on retinal ganglion cells in larval but not juvenile zebrafish. LTP induction requires the activation of postsynaptic N-methyl-D-aspartate receptors, and its expression involves arachidonic acid-dependent presynaptic changes in calcium dynamics and neurotransmitter release. Physiologically, both electrical and visual stimulation-induced LTP can enhance visual responses of retinal ganglion cells. Thus, LTP exists in developing retinae with a presynaptic locus and may serve for visual experience-dependent refinement of retinal circuits.

Inhibition of phospholipase A1, lipase and galactolipase activities of pancreatic lipase-related protein 2 by methyl arachidonyl fluorophosphonate (MAFP)

Methyl arachidonyl fluorophosphonate (MAFP) is a known inhibitor of cytosolic phospholipase A2 and some other serine enzymes. MAFP was found here to be an irreversible inhibitor of human pancreatic lipase-related protein 2 (HPLRP2), an enzyme displaying lipase, phospholipase A1 and galactolipase activities. In the presence of MAFP, mass spectrometry analysis of HPLRP2 revealed a mass increase of 351Da, suggesting a covalent binding of MAFP to the active site serine residue. When HPLRP2 was pre-incubated with MAFP before measuring residual activity, a direct inhibition of HPLRP2 occurred, confirming that HPLRP2 has an active site freely accessible to solvent and differs from most lipases in solution. HPLRP2 activities on tributyrin (TC4), phosphatidylcholine (PC) and monogalactosyl dioctanoylglycerol (C8-MGDG) were equally inhibited under these conditions. Bile salts were not required to trigger the inhibition, but they significantly increased the rate of HPLRP2 inhibition, probably because of MAFP micellar solubilization. Since HPLRP2 is active on various substrates that self-organize differently in the presence of water, HPLRP2 inhibition by MAFP was tested in the presence of these substrates after adding MAFP in the course of the lipolysis reaction. In this case, the rates of inhibition of lipase, phospholipase A1 and galactolipase activities were not equivalent (triglycerides>PC>MGDG), suggesting different enzyme/inhibitor partitioning between the aqueous phase and lipid aggregates. The inhibition by MAFP of a well identified phospholipase A1 (HPLRP2), present in pancreatic juice and also in human monocytes, indicates that MAFP cannot be used for discriminating phospholipase A2 from A1 activities at the cellular level.

A novel C(28)-hydroxylated lupeolic acid suppresses the biosynthesis of eicosanoids through inhibition of cytosolic phospholipase A2

Eicosanoids are potent lipid mediators derived from phospholipase (PL)-released arachidonic acid (AA) coupled to subsequent metabolism by cyclooxygenase (COX)-1/2 or lipoxygenases (LO) which are involved in a variety of homeostatic biological functions and inflammation. We have investigated three lupeolic acids (LA) from the gum resin of Boswellia carterii for their ability to interfere with eicosanoid biosynthesis in human blood cells. A novel, yet unknown C(28)-hydroxylated LA, that is, 3α-acetoxy-28-hydroxylup-20(29)-en-4β-oic acid (Ac-OH-LA) was found to inhibit the biosynthesis of COX-, 5-LO- and 12-LO-derived eicosanoids from endogenous AA in activated platelets, neutrophils, and monocytes from human blood with consistent IC50 values of 2.3–6.9μM. In contrast, two other LAs lacking the C(28)-OH moiety were essentially inactive in this respect. Inhibition of eicosanoids by Ac-OH-LA correlated with reduced release of AA in intact cells. When AA was exogenously provided as substrate for cellular eicosanoid biosynthesis the inhibitory effects of Ac-OH-LA were essentially reversed, even though some inhibition of 5-LO and COX-1 product formation still remained. Finally, by means of a cell-free phospholipid hydrolysis assay using human recombinant cytosolic PLA2α, we show that Ac-OH-LA may directly interfere with cPLA2α activity (IC50=3.6μM). Together, we identified a novel, naturally occuring C(28)-hydroxylated LA which acts as efficient inhibitor of cPLA2α and consequently suppresses eicosanoid biosynthesis in intact cells.