RESUMO
Spinal cord injury (SCI) results in permanent loss of motor functions. A significant aspect of the tissue damage and functional loss may be preventable as it occurs, secondary to the trauma. We show that the phospholipase A(2) (PLA(2)) superfamily plays important roles in SCI. PLA(2) enzymes hydrolyze membrane glycerophospholipids to yield a free fatty acid and lysophospholipid. Some free fatty acids (arachidonic acid) give rise to eicosanoids that promote inflammation, while some lysophospholipids (lysophosphatidylcholine) cause demyelination. We show in a mouse model of SCI that two cytosolic forms [calcium-dependent PLA(2) group IVA (cPLA(2) GIVA) and calcium-independent PLA(2) group VIA (iPLA(2) GVIA)], and a secreted form [secreted PLA(2) group IIA (sPLA(2) GIIA)] are up-regulated. Using selective inhibitors and null mice, we show that these PLA(2)s play differing roles. cPLA(2) GIVA mediates protection, whereas sPLA(2) GIIA and, to a lesser extent, iPLA(2) GVIA are detrimental. Furthermore, completely blocking all three PLA(2)s worsens outcome, while the most beneficial effects are seen by partial inhibition of all three. The partial inhibitor enhances expression of cPLA(2) and mediates its beneficial effects via the prostaglandin EP1 receptor. These findings indicate that drugs that inhibit detrimental forms of PLA(2) (sPLA(2) and iPLA2) and up-regulate the protective form (cPLA2) may be useful for the treatment of SCI.
Assuntos
Fosfolipases A2/metabolismo , Traumatismos da Medula Espinal/enzimologia , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Feminino , Fosfolipases A2 do Grupo II/antagonistas & inibidores , Fosfolipases A2 do Grupo II/deficiência , Fosfolipases A2 do Grupo II/metabolismo , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Fosfolipases A2 do Grupo IV/deficiência , Fosfolipases A2 do Grupo IV/genética , Fosfolipases A2 do Grupo IV/metabolismo , Fosfolipases A2 do Grupo VI/antagonistas & inibidores , Fosfolipases A2 do Grupo VI/deficiência , Fosfolipases A2 do Grupo VI/metabolismo , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Inibidores de Fosfolipase A2 , Fosfolipases A2/classificação , Fosfolipases A2/deficiência , Receptor Cross-Talk , Receptores de Prostaglandina E Subtipo EP1/metabolismo , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
The phospholipase A(2) (PLA(2)) superfamily hydrolyzes phospholipids to release free fatty acids and lysophospholipids, some of which can mediate inflammation and demyelination, hallmarks of the CNS autoimmune disease multiple sclerosis. The expression of two of the intracellular PLA(2)s (cPLA(2) GIVA and iPLA(2) GVIA) and two of the secreted PLA(2)s (sPLA(2) GIIA and sPLA(2) GV) are increased in different stages of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We show using small molecule inhibitors, that cPLA(2) GIVA plays a role in the onset, and iPLA(2) GVIA in the onset and progression of EAE. We also show a potential role for sPLA(2) in the later remission phase. These studies demonstrate that selective inhibition of iPLA(2) can ameliorate disease progression when treatment is started before or after the onset of symptoms. The effects of these inhibitors on lesion burden, chemokine and cytokine expression as well as on the lipid profile provide insights into their potential modes of action. iPLA(2) is also expressed by macrophages and other immune cells in multiple sclerosis lesions. Our results therefore suggest that iPLA(2) might be an excellent target to block for the treatment of CNS autoimmune diseases, such as multiple sclerosis.
Assuntos
Encefalomielite Autoimune Experimental/enzimologia , Fosfolipases A2/fisiologia , Isoformas de Proteínas/fisiologia , Medula Espinal/enzimologia , Adulto , Amidas/uso terapêutico , Animais , Citocinas/imunologia , Progressão da Doença , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/genética , Inibidores Enzimáticos/uso terapêutico , Ácidos Graxos/análise , Feminino , Citometria de Fluxo , Imunofluorescência , Fluorocarbonos , Expressão Gênica , Humanos , Imuno-Histoquímica , Cetonas/uso terapêutico , Macrófagos/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/enzimologia , Esclerose Múltipla/genética , Inibidores de Fosfolipase A2 , Fosfolipases A2/química , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , RNA Mensageiro/análise , Linfócitos T/enzimologia , Adulto JovemRESUMO
There is increasing evidence for a major and critical involvement of lipids in signal transduction and cellular trafficking, and this has motivated large-scale studies on lipid pathways. The Lipid Metabolites and Pathways Strategy consortium is actively investigating lipid metabolism in mammalian cells and has made available time-course data on various lipids in response to treatment with KDO(2)-lipid A (a lipopolysaccharide analog) of macrophage RAW 264.7 cells. The lipids known as eicosanoids play an important role in inflammation. We have reconstructed an integrated network of eicosanoid metabolism and signaling based on the KEGG pathway database and the literature and have developed a kinetic model. A matrix-based approach was used to estimate the rate constants from experimental data and these were further refined using generalized constrained nonlinear optimization. The resulting model fits the experimental data well for all species, and simulated enzyme activities were similar to their literature values. The quantitative model for eicosanoid metabolism that we have developed can be used to design experimental studies utilizing genetic and pharmacological perturbations to probe fluxes in lipid pathways.
Assuntos
Eicosanoides/química , Modelos Químicos , Algoritmos , Simulação por Computador , Cinética , Dinâmica não Linear , Reprodutibilidade dos Testes , Transdução de SinaisRESUMO
A series of 2-oxoamides based on dipeptides and pseudodipeptides were synthesized and their activities towards two human intracellular phospholipases A(2) (GIVA cPLA(2) and GVIA iPLA(2)) and one human secretory phospholipase A(2) (GV sPLA(2)) were evaluated. Derivatives containing a free carboxyl group are selective GIVA cPLA(2) inhibitors. A derivative based on the ethyl ester of an ether pseudodipeptide is the first 2-oxoamide, which preferentially inhibits GVIA iPLA(2). The effect of 2-oxoamides on the generation of arachidonic acid from RAW 264.7 macrophages was also studied and it was found that selective GIVA cPLA(2) inhibitors preferentially inhibited cellular arachidonic acid release; one pseudodipeptide gave an IC(50) value of 2muM.
Assuntos
Dipeptídeos/química , Fosfolipases A2 Citosólicas/antagonistas & inibidores , Fosfolipases A2 Secretórias/antagonistas & inibidores , Piridinas/química , Piridinas/farmacologia , Animais , Ácido Araquidônico/metabolismo , Linhagem Celular Tumoral , Humanos , Concentração Inibidora 50 , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Fosfolipases A2 Citosólicas/metabolismo , Fosfolipases A2 Secretórias/metabolismo , Piridinas/síntese química , Relação Estrutura-AtividadeRESUMO
We provide evidence that two members of the intracellular phospholipase A(2) family, namely calcium-dependent group IVA (cPLA(2) GIVA) and calcium-independent group VIA (iPLA(2) GVIA) may play important roles in Wallerian degeneration in the mouse sciatic nerve. We assessed the roles of these PLA(2)s in cPLA(2) GIVA(-/-) mice, and mice treated with a selective inhibitor of iPLA(2) GVIA (FKGK11). Additionally, the effects of both these PLA(2)s were assessed by treating cPLA(2) GIVA(-/-) mice with the iPLA(2) inhibitor. Our data suggest that iPLA(2) GVIA may play more of a role in the early stages of myelin breakdown, while cPLA(2) GIVA may play a greater role in myelin clearance by macrophages. Our results also show that the delayed myelin clearance and Wallerian degeneration after sciatic nerve crush injury in mice lacking cPLA(2) and iPLA(2) activities is accompanied by a delay in axon regeneration, target re-innervation and functional recovery. These results indicate that the intracellular PLA(2)s (cPLA(2) GIVA and iPLA(2) GVIA) contribute significantly to various aspects of Wallerian degeneration in injured peripheral nerves, which is then essential for successful axon regeneration. This work has implications for injury responses and recovery after peripheral nerve injuries in humans, as well as for understanding the slow clearance of myelin after CNS injury and its potential consequences for axon regeneration.
Assuntos
Axônios/fisiologia , Fosfolipases A2 do Grupo IV/metabolismo , Fosfolipases A2 do Grupo VI/metabolismo , Regeneração Nervosa/fisiologia , Degeneração Walleriana/enzimologia , Animais , Axônios/enzimologia , Imunofluorescência , Fluorocarbonos , Fosfolipases A2 do Grupo IV/genética , Fosfolipases A2 do Grupo VI/antagonistas & inibidores , Fosfolipases A2 do Grupo VI/genética , Cetonas/farmacologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Modelos Animais , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nervo Isquiático/enzimologia , Nervo Isquiático/lesões , Nervo Isquiático/patologia , Pele/inervação , Degeneração Walleriana/patologiaRESUMO
The Lipid Metabolites and Pathway Strategy (LIPID MAPS) Consortium is a nationwide initiative that has taken on the task of employing lipidomics to advance our understanding of lipid metabolism at the molecular and mechanistic level in living organisms. An important step toward this goal is to craft enabling analytical procedures to comprehensively measure all lipid species, to establish the precise structural identity of the lipid molecules analyzed, and to generate accurate quantitative information. The LIPID MAPS Consortium has succeeded in the implementation of a complete infrastructure that now provides tools for analysis of the global lipidome in cultured and primary cells. Here we illustrate the advancement of a gas chromatography mass spectrometry (GC/MS) procedure for the analysis of essential fatty acids in RAW 264.7 cells. Our method allows for the specific identification and quantification of over 30 fatty acids present in cells in their free form in a single analytical GC/MS run. Free fatty acids are selectively extracted in the presence of deuterated internal standards, which permit subsequent estimation of extraction efficiencies and quantification with high accuracy. Mass spectrometer conditions were optimized for single-ion monitoring, which provides an extremely sensitive technology to measure fatty acids from biological samples in trace amounts. These methods will be presented in the context of our broader effort to analyze all fatty acids as well as their metabolites in inflammatory cells.
Assuntos
Ácidos Graxos Essenciais/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Macrófagos/química , Animais , Linhagem Celular Tumoral , Camundongos , Reprodutibilidade dos TestesRESUMO
A variety of 2-oxoamides and related amides based on natural and non-natural amino acids were synthesized. Their activity on two human intracellular phospholipases (GIVA cPLA(2) and GVIA iPLA(2)) and one human secretory phospholipase (GV sPLA(2)) was evaluated. We show that an amide based on (R)-gamma-norleucine is a highly selective inhibitor of GV sPLA(2).
Assuntos
Aminoácidos/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores de Fosfolipase A2 , Piridinas/química , Amidas/química , Inibidores Enzimáticos/síntese química , Humanos , Estrutura Molecular , Fosfolipases A2/farmacologia , Relação Estrutura-AtividadeRESUMO
The Group IVA cytosolic phospholipase A2 (GIVA cPLA2) is a key provider of substrates for the production of eicosanoids and platelet-activating factor. We explored the structure-activity relationship of 2-oxoamide-based compounds and GIVA cPLA2 inhibition. The most potent inhibitors are derived from delta- and gamma-amino acid-based 2-oxoamides. The optimal side-chain moiety is a short nonpolar aliphatic chain. All of the newly developed 2-oxoamides as well as those previously described have now been tested with the human Group V secreted PLA2 (GV sPLA2) and the human Group VIA calcium-independent PLA2 (GVIA iPLA2). Only one 2-oxoamide compound had appreciable inhibition of GV sPLA2, and none of the potent GIVA cPLA2 inhibitors inhibited either GV sPLA2 or GVIA iPLA2. Two of these specific GIVA cPLA2 inhibitors were also found to have potent therapeutic effects in animal models of pain and inflammation at dosages well below the control nonsteroidal anti-inflammatory drugs.
Assuntos
Amidas/síntese química , Aminoácidos/síntese química , Anti-Inflamatórios não Esteroides/síntese química , Fosfolipases A/antagonistas & inibidores , Amidas/química , Amidas/farmacologia , Aminoácidos/química , Aminoácidos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Carragenina , Edema/induzido quimicamente , Edema/tratamento farmacológico , Fosfolipases A2 do Grupo IV , Fosfolipases A2 do Grupo V , Fosfolipases A2 do Grupo VI , Humanos , Inflamação/tratamento farmacológico , Dor/tratamento farmacológico , Fosfolipases A2 , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Inhibitors of the Group IVA phospholipase A(2) (GIVA cPLA(2)) and GVIA iPLA(2) are useful tools for defining the roles of these enzymes in cellular signaling and inflammation. We have developed inhibitors of GVIA iPLA(2) building upon the 2-oxoamide backbone that are uncharged, containing ester groups. Although the most potent inhibitors of GVIA iPLA(2) also inhibited GIVA cPLA(2), there were three 2-oxoamide compounds that selectively and weakly inhibited GVIA iPLA(2). We further show that several potent 2-oxoamide inhibitors of GIVA cPLA(2) containing free carboxylic groups (Kokotos et al. J. Med. Chem. 2002, 45, 2891-2893) do not inhibit GVIA iPLA(2) and are, therefore, selective GIVA cPLA(2) inhibitors.
Assuntos
Fosfolipases A/antagonistas & inibidores , Piridinas/química , Piridinas/farmacologia , Animais , Linhagem Celular , Dinoprostona/biossíntese , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Camundongos , Estrutura Molecular , Fosfolipases A/classificação , Fosfolipases A/metabolismo , Piridinas/síntese química , Relação Estrutura-AtividadeRESUMO
A variety of lipophilic 2-oxoamides containing sulfonamide analogs of gamma-amino acids as well as acyl sulfonamides of gamma-aminobutyric acid were synthesized. Their ability to inhibit intracellular GIVA cPLA2 and GVIA iPLA2 as well as secreted GV sPLA2 was evaluated. The sulfonamide group seems a bioisosteric group suitable to replace the carboxyl group in 2-oxoamide inhibitors of GVIA cPLA2.
Assuntos
Aminoácidos/química , Aminoácidos/fisiologia , Fosfolipases A2/metabolismo , Piridinas/síntese química , Sulfonamidas/química , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Inibidores de Fosfolipase A2 , Piridinas/farmacologia , Sulfonamidas/farmacologiaRESUMO
The development of selective inhibitors for individual PLA(2) enzymes is necessary in order to target PLA(2)-specific signaling pathways, but it is challenging due to the observed promiscuity of known PLA(2) inhibitors. In the current work, we present the development and application of a variety of synthetic routes to produce pentafluoro, tetrafluoro, and trifluoro derivatives of activated carbonyl groups in order to screen for selective inhibitors and characterize the chemical properties that can lead to selective inhibition. Our results demonstrate that the pentafluoroethyl ketone functionality favors selective inhibition of the GVIA iPLA(2), a very important enzyme for which specific, potent, reversible inhibitors are needed. We find that 1,1,1,2,2-pentafluoro-7-phenyl-heptan-3-one (FKGK11) is a selective inhibitor of GVIA iPLA(2) (X(I)(50) = 0.0073). Furthermore, we conclude that the introduction of an additional fluorine atom at the alpha' position of a trifluoromethyl ketone constitutes an important strategy for the development of new potent GVIA iPLA(2) inhibitors.
Assuntos
Inibidores Enzimáticos/síntese química , Cetonas/síntese química , Fosfolipases A2 Citosólicas/química , Carbono/química , Química Farmacêutica/métodos , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Fluorocarbonos , Humanos , Cetonas/química , Cetonas/farmacologia , Espectroscopia de Ressonância Magnética , Micelas , Modelos Químicos , Fosfolipases A2 Citosólicas/antagonistas & inibidores , Fosfolipídeos/química , Transdução de Sinais , Especificidade por SubstratoRESUMO
The GIVA phospholipase A(2) (PLA(2)) contains two domains: a calcium-binding domain (C2) and a catalytic domain. These domains are linked via a flexible tether. GIVA PLA(2) activity is Ca(2+)-dependent in that calcium binding promotes protein docking to the phospholipid membrane. In addition, the catalytic domain has a lid that covers the active site, presumably regulating GIVA PLA(2) activity. We now present studies that explore the dynamics and conformational changes of this enzyme in solution utilizing peptide amide hydrogen/deuterium (H/D) exchange coupled with liquid chromatography-mass spectrometry (DXMS) to probe the solvent accessibility and backbone flexibility of the C2 domain, the catalytic domain, and the intact GIVA PLA(2). We also analyzed the changes in H/D exchange of the intact GIVA PLA(2) upon Ca(2+) binding. The DXMS results showed a fast H/D-exchanging lid and a slow exchanging central core. The C2 domain showed two distinct regions: a fast exchanging region facing away from the catalytic domain and a slow exchanging region present in the "cleft" region between the C2 and catalytic domains. The slow exchanging region of the C2 domain is in tight proximity to the catalytic domain. The effects of Ca(2+) binding on GIVA PLA(2) are localized in the C2 domain and suggest that binding of two distinct Ca(2+) ions causes tightening up of the regions that surround the anion hole at the tip of the C2 domain. This conformational change may be the initial step in GIVA PLA(2) activation.
Assuntos
Cálcio/química , Fosfolipases A2/química , Animais , Sítios de Ligação/fisiologia , Cálcio/metabolismo , Membrana Celular/química , Membrana Celular/enzimologia , Cromatografia Líquida , Deutério/química , Ativação Enzimática/fisiologia , Humanos , Hidrogênio/química , Espectrometria de Massas , Fosfolipases A2/metabolismo , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologiaRESUMO
A variety of lipophilic 2-oxoamides based on gamma-aminobutyric and delta-aminovaleric analogues were synthesized. 2-oxoamides containing a tetrazole, a thioethyl or a thioacetyl group are weak inhibitors of GIVA cPLA(2), while derivatives containing a methyl tetrazole, a diethyl phosphonate or a thioethyl group are weak inhibitors of GV sPLA(2).
Assuntos
Amidas/síntese química , Aminoácidos Neutros/química , Inibidores de Fosfolipase A2 , Ácido gama-Aminobutírico/química , Ácidos Palmíticos/química , Fosfolipases A2/metabolismo , Valeratos/química , Ácido gama-Aminobutírico/análogos & derivadosRESUMO
Arachidonic acid is released by phospholipase A(2) and converted into hundreds of distinct bioactive mediators by a variety of cyclooxygenases (COX), lipoxygenases (LO), and cytochrome P450s. Because of the size and diversity of the eicosanoid class of signaling molecules produced, a thorough and systematic investigation of these biological processes requires the simultaneous quantitation of a large number of eicosanoids in a single analysis. We have developed a robust liquid chromatography/tandem mass spectrometry method that can identify and quantitate over 60 different eicosanoids in a single analysis, and we applied it to agonist-stimulated RAW264.7 murine macrophages. Fifteen different eicosanoids produced through COX and 5-LO were detected either intracellularly or in the media following stimulation with 16 different agonists of Toll-like receptors (TLR), G protein-coupled receptors, and purinergic receptors. No significant differences in the COX metabolite profiles were detected using the different agonists; however, we determined that only agonists creating a sustained Ca(2+) influx were capable of activating the 5-LO pathway in these cells. Synergy between Ca(2+) and TLR pathways was detected and discovered to be independent of NF-kappaB-induced protein synthesis. This demonstrates that TLR induction of protein synthesis and priming for enhanced phospholipase A(2)-mediated eicosanoid production work through two distinct pathways.
Assuntos
Cálcio/metabolismo , Eicosanoides/metabolismo , Receptor 4 Toll-Like/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Lipídeos/química , Camundongos , Modelos Biológicos , NF-kappa B/metabolismo , Fosfolipases A/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Fatores de TempoRESUMO
Phospholipase A(2) (PLA(2)) forms are expressed in spinal cord, and inhibiting spinal PLA(2) induces a potent antihyperalgesia. Here, we examined the antihyperalgesic effects after systemic and i.t. delivery of four compounds constructed with a common motif consisting of a 2-oxoamide with a hydrocarbon tail and a four-carbon tether. These molecules were characterized for their ability to block group IVA calcium-dependent PLA(2) (cPLA(2)) and group VIA calcium-independent PLA(2) (iPLA(2)) in inhibition assays using human recombinant enzyme. The rank ordering of potency in blocking group IVA cPLA(2) was AX048 (ethyl 4-[(2-oxohexadecanoyl)amino]butanoate), AX006 (4-[(2-oxohexadecanoyl)amino]butanoic acid), and AX057 (tert-butyl 4-[(2-oxohexadecanoyl)amino]butanoate) > AX010 (methyl 4-[(2-oxohexadecanoyl)amino]butanoate) and for inhibiting group VIA iPLA(2) was AX048, AX057 > AX006, and AX010. No agent altered recombinant cyclooxygenase activity. In vivo, i.t. (30 mug) and systemic (0.2-3 mg/kg i.p.) AX048 blocked carrageenan hyperalgesia and after systemic delivery in a model of spinally mediated hyperalgesia induced by i.t. substance P (SP). The other agents were without activity. In rats prepared with lumbar i.t. loop dialysis catheters, SP evoked spinal prostaglandin E(2) (PGE(2)) release. AX048 alone inhibited PGE(2) release. Intrathecal SR141617, a cannabinoid CB1 inhibitor at doses that blocked the effects of i.t. anandamide had no effect upon i.t. AX048. These results suggest that AX048 is the first systemically bioavailable compound with a significant affinity for group IVA cPLA(2), which produces a potent antihyperalgesia. The other agents, although demonstrating enzymatic activity in cell-free assays, appear unable to gain access to the intracellular PLA(2) toward which their action is targeted.
Assuntos
Dinoprostona/metabolismo , Inibidores Enzimáticos/farmacologia , Hiperalgesia/tratamento farmacológico , Fosfolipases A/antagonistas & inibidores , Medula Espinal/metabolismo , Ácido gama-Aminobutírico/análogos & derivados , Animais , Carragenina , Fenômenos Químicos , Físico-Química , Inibidores de Ciclo-Oxigenase/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/efeitos adversos , Temperatura Alta , Hiperalgesia/induzido quimicamente , Injeções Intraperitoneais , Injeções Espinhais , Masculino , Microdiálise , Oxirredução , Fosfolipases A/metabolismo , Fosfolipases A2 , Ratos , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/antagonistas & inibidores , Medula Espinal/efeitos dos fármacos , Medula Espinal/enzimologia , Substância P , Ácido gama-Aminobutírico/administração & dosagem , Ácido gama-Aminobutírico/efeitos adversos , Ácido gama-Aminobutírico/farmacologiaRESUMO
P388D1 cells release arachidonic acid (AA) and produce prostaglandin E2 (PGE2) upon long-term stimulation with lipopolysaccharide (LPS). The cytosolic Group IVA (GIVA) phospholipase A2 (PLA2) has been implicated in this pathway. LPS stimulation also results in increased expression and secretion of a secretory PLA2, specifically GV PLA2. To test whether GV PLA2 contributes to PGE2 production and whether GIVA PLA2 activation increases the expression of GV PLA2, we utilized the specific GIVA PLA2 inhibitor pyrrophenone and second generation antisense oligonucleotides (AS-ONs) designed to specifically inhibit expression and activity of GV PLA2. Treatment of P388D1 cells with antisense caused a marked decrease in basal GV PLA2 mRNA and prevented the LPS-induced increase in GV PLA2 mRNA. LPS-stimulated cells release active GV PLA2 into the medium, which is inhibited to background levels by antisense treatment. However, LPS-induced PGE2 release by antisense-treated cells and by control cells are not significantly different. Collectively, the results suggest that the upregulation of GV PLA2 during long-term LPS stimulation is not required for PGE2 production by P388D1 cells. Experiments employing pyrrophenone suggested that GIVA PLA2 is the dominant player involved in AA release, but it appears not to be involved in the regulation of LPS-induced expression of GV PLA2 or cyclooxygenase-2.
Assuntos
Dinoprostona/fisiologia , Lipopolissacarídeos/metabolismo , Fosfolipases A/metabolismo , Regulação para Cima , Animais , Ácido Araquidônico/metabolismo , Linhagem Celular , Meios de Cultura/metabolismo , Ciclo-Oxigenase 2/metabolismo , DNA Complementar/metabolismo , Dinoprostona/metabolismo , Eicosanoides/metabolismo , Inibidores Enzimáticos/farmacologia , Fosfolipases A2 do Grupo V , Immunoblotting , Indóis/farmacologia , Lipídeos/química , Macrófagos/metabolismo , Camundongos , Oligonucleotídeos Antissenso/química , Fosfolipases A2 , Pirrolidinas/química , RNA/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , TransfecçãoRESUMO
OmpT is a surface protease of gram-negative bacteria that has been shown to cleave antimicrobial peptides, activate human plasminogen, and degrade some recombinant heterologous proteins. We have analyzed the substrate specificity of OmpT by two complementary substrate filamentous phage display methods: (i) in situ cleavage of phage that display protease-susceptible peptides by Escherichia coli expressing OmpT and (ii) in vitro cleavage of phage-displayed peptides using purified enzyme. Consistent with previous reports, OmpT was found to exhibit a virtual requirement for Arg in the P1 position and a slightly less stringent preference for this residue in the P1' position (P1 and P1' are the residues immediately prior to and following the scissile bond). Lys, Gly, and Val were also found in the P1' position. The most common residues in the P2' position were Val or Ala, and the P3 and P4 positions exhibited a preference for Trp or Arg. Synthetic peptides based upon sequences selected by bacteriophage display were cleaved very efficiently, with kcat/Km values up to 7.3 x 10(6) M(-1) s(-1). In contrast, a peptide corresponding to the cleavage site of human plasminogen was hydrolyzed with a kcat/Km almost 10(6)-fold lower. Overall, the results presented in this work indicate that in addition to the P1 and P1' positions, additional amino acids within a six-residue window (between P4 and P2') contribute to the binding of substrate polypeptides to the OmpT binding site.