RESUMO
The goal of the current study was to examine the formation of phospholipids, 1-radyl-2-lysosn-glycero-phospholipids (lyso-PL) and 2-acetylated phospholipids (such as PAF) as well as mechanisms responsible for generating these phospholipids in bronchoalveolar lavage fluid (BAI.F) from allergic subjects challenged with antigen. Bronchoalveolar lavage was performed in normal and allergic subjects before, 5-30 min, 6 h, and 20 h after segmental antigen challenge via a wedged bronchoscope. Levels of 1-hexadecyl-2-lyso-phospholipids and 1-hexadecyl-2-acetyl-phospholipids were initially determined by negative ion chemical ionization gas chromatography/mass spectrometry (NICI-GC/MS). Antigen dramatically elevated quantities of 1-hexadecyl-2-lyso-phospholipids in allergic subjects 20 h after challenge when compared to non-allergic controls. In contrast, there was not a significant increase in levels of 1-hexadecyl-2-acetyl-phospholipids after antigen challenge. Closer examination of 1-radyl-2-lyso-sn-glycero-3-phosphocholine (GPC) revealed that 1-palmitoyl-2-lyso-GPC, 1-myristoyl-2-lyso-GPC and 1-hexadecyl-2-lyso-GPC were three major molecular species produced after antigen challenge. 1-palmitoyl-2-lyso-GPC increased sevenfold to levels of 222 +/- 75 ng/ml of BALF 20 h after antigen challenge. The elevated levels of lyso-PL correlated with levels of albumin used to assess plasma exudation induced by allergen challenge. In contrast, the time course of prostaglandin D2 (PGD2) or 9 alpha, 11 beta PGF2 (11 beta PGF2) formation did not correlate with lyso-PL generation. To examine the mechanism leading to lyso-phospholipid formation in antigen-challenged allergic subjects, secretory phospholipase A2 (PI.A2) and acetyl hydrolase activities were measured. There was a significant increase in PLA2 activity found in BALF of allergic subjects challenged with antigen when compared to saline controls. This activity was neutralized by an antibody directed against low molecular mass, (14 kD) human synovial PLA2 and dithiothreitol. Acetyl hydrolase activity also markedly increased in BALF obtained after antigen challenge. This study indicates that high levels of lyso-PLs are present in airways of allergic subjects challenged with antigen and provides evidence for two distinct mechanisms that could induce lyso-PL formation. Future studies will be necessary to determine the ramifications of these high levels of lyso-phospholipids on airway function.
Assuntos
Asma/fisiopatologia , Líquido da Lavagem Broncoalveolar/química , Hipersensibilidade/fisiopatologia , Lisofosfolipídeos/biossíntese , Cloreto de Metacolina/imunologia , Rinite/fisiopatologia , Adulto , Asma/complicações , Lavagem Broncoalveolar , Dinoprosta/análise , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Hipersensibilidade/complicações , Lisofosfolipídeos/análise , Masculino , Prostaglandina D2/análise , Valores de Referência , Rinite/complicações , Fatores de TempoRESUMO
Non-invasive biomarkers will enable widespread screening and early diagnosis of Alzheimer's disease (AD). We hypothesized that the considerable loss of brain tissue in AD will result in detection of brain lipid components in urine, and that these will change in concert with CSF and brain biomarkers of AD. We examined urine dicarboxylic acids (DCA) of carbon length 3-10 to reflect products of oxidative damage and energy generation or balance that may account for changes in brain function in AD. Mean C4-C5 DCAs were lower and mean C7-C10 DCAs were higher in the urine from AD compared to cognitively healthy (CH) individuals. Moreover, mean C4-C5 DCAs were lower and mean C7-C9 were higher in urine from CH individuals with abnormal compared to normal CSF amyloid and Tau levels; i.e., the apparent urine changes in AD also appeared to be present in CH individuals that have CSF risk factors of early AD pathology. In examining the relationship between urine DCAs and AD biomarkers, we found short chain DCAs positively correlated with CSF Aß42, while C7-C10 DCAs negatively correlated with CSF Aß42 and positively correlated with CSF Tau levels. Furthermore, we found a negative correlation of C7-C10 DCAs with hippocampal volume (p < 0.01), which was not found in the occipital volume. Urine measures of DCAs have an 82% ability to predict cognitively healthy participants with normal CSF amyloid/Tau. These data suggest that urine measures of increased lipoxidation and dysfunctional energy balance reflect early AD pathology from brain and CSF biomarkers. Measures of urine DCAs may contribute to personalized healthcare by indicating AD pathology and may be utilized to explore population wellness or monitor the efficacy of therapies in clinical trials.
Assuntos
Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Ácidos Dicarboxílicos/urina , Hipocampo/patologia , Proteínas tau/líquido cefalorraquidiano , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/patologia , Doenças Assintomáticas , Biomarcadores/líquido cefalorraquidiano , Biomarcadores/urina , Disfunção Cognitiva/diagnóstico , Disfunção Cognitiva/diagnóstico por imagem , Ácidos Dicarboxílicos/química , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Imageamento por Ressonância Magnética , Masculino , Análise Multivariada , Fatores de RiscoRESUMO
The current study evaluates the capacity of recombinant rat stem cell factor (rrSCF) to regulate enzymes that control AA release and eicosanoid generation in mouse bone marrow-derived mast cells (BMMCs). Initial studies indicated that rrSCF provided for 24 h inhibited the release of AA into supernatant fluids of antigen- and ionophore A23187-stimulated BMMCs. Agonist-induced increases in cellular levels of AA were also inhibited, albeit to a lesser degree by rrSCF. To determine the inhibitory mechanism, several steps (e.g., mobilization of cytosolic calcium, release of BMMC granules, and regulation of phospholipase A2 [PLA2] activity) that could influence AA release were measured in rrSCF-treated cells. rrSCF did not alter the capacity of BMMCs to mobilize cytosolic calcium or release histamine in response to antigen and ionophore. BMMCs released large amounts of PLA2 with characteristics of the group II family in response to antigen and ionophore A23187. rrSCF treatment of BMMCs reduced the secretion of this PLA2 activity by BMMCs. Partial purification of acid-extractable PLA2 from rrSCF-treated and untreated BMMCs suggested that rrSCF decreased the quantity of acid-stable PLA2 within the cells. In contrast to group II PLA2, the quantity of cPLA2 (as determined by Western blot analysis) increased in response to rrSCF. To assess the ramifications of rrSCF-induced reductions in AA and group II PLA2, eicosanoid formation was measured in antigen- and ionophore-stimulated BMMCs, rrSCF-inhibited (100 ng/ml, 24 h) prostaglandin D2 (PGD2), thromboxane B2, and leukotriene B4 by 48.4 +/- 7.7%, 61.1 +/- 10.0% AND 38.1 +/- 3.6%, respectively, in antigen-stimulated cells. Similar patterns of inhibition were observed in ionophore-stimulated BMMCs. The addition of a group I PLA2 or exogenous AA to BMMCs reversed the inhibition of eicosanoid generation induced by rrSCF. Together, these data indicate that rrSCF differentially regulates group II and cytosolic PLA2 activities in BMMCs. The resultant reductions in eicosanoid generation suggest that group II PLA2 provides a portion of AA that is used for eicosanoid biosynthesis by BMMCs.
Assuntos
Ácido Araquidônico/metabolismo , Eicosanoides/metabolismo , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Mastócitos/metabolismo , Fosfolipases A/metabolismo , Animais , Calcimicina/farmacologia , Cálcio/metabolismo , Células Cultivadas , Citosol/metabolismo , Relação Dose-Resposta a Droga , Ácidos Graxos não Esterificados/metabolismo , Cinética , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos CBA , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Ratos , Proteínas Recombinantes/farmacologia , Fator de Células-Tronco , Fatores de TempoRESUMO
Healthy volunteers supplemented their usual Western diets with Promega fish oil supplement (eicosapentaenoic acid [EPA], 0.28 g; docosahexaenoic acid [DCHA], 0.12 g; other n-3 fatty acids 0.10 g per capsule) using three protocols. Initial experiments (protocol 1 and 2) investigated the kinetics of incorporation of n-3 fatty acids into serum and neutrophil lipids after 10 capsules/d of Promega. EPA was rapidly detected in both serum and neutrophil lipids; the arachidonic acid (AA) to EPA ratio in neutrophil phospholipids showed a maximal reduction of 49:1 to 8:1 within 1 wk of beginning supplementation. EPA was preferentially incorporated into phosphatidyl-ethanolamine and phosphatidylcholine but not phosphatidylinositol. Long-term supplementation for up to 7 wk did not influence the AA/EPA ratio or the distribution of EPA among neutrophil phospholipids in a manner that was not observed after the first week. Neutrophils produced similar quantities of platelet-activating factor and slightly lower quantities of leukotriene B4 during long-term supplementation when compared with presupplementation values. Experiments examining the influence of Promega dosage indicated that the AA/EPA ratio in neutrophil lipids decreased in a dose-dependent manner. Only when the dose was increased to 15 capsules/d was there a reduction in the AA/DCHA ratio in neutrophil lipids. The quantity of AA in neutrophil lipids remained relatively constant at all supplement doses. Taken together, the current study demonstrates the capacity of n-3 fatty acids provided with a Western diet to be rapidly incorporated into neutrophil lipids. However, dietary n-3 fatty acids appear not to significantly reduce arachidonate content within neutrophil phospholipids. Constant arachidonate levels may account for the lack of large reductions in the biosynthesis of lipid mediators by neutrophils after fish-oil supplementation.
Assuntos
Gorduras na Dieta/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Óleos de Peixe/farmacologia , Lipídeos/sangue , Neutrófilos/metabolismo , Fosfolipídeos/sangue , Adulto , Relação Dose-Resposta a Droga , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/biossíntese , Ácido Eicosapentaenoico/sangue , Ácidos Graxos/sangue , Humanos , Cinética , Leucotrieno B4/biossíntese , Leucotrieno B4/sangue , Masculino , Neutrófilos/efeitos dos fármacos , Fosfolipídeos/isolamento & purificação , Fator de Ativação de Plaquetas/biossíntese , Valores de Referência , Fatores de TempoRESUMO
Recent studies have demonstrated that arachidonic acid (AA) may serve as an important signal that blocks cell proliferation of certain neoplastic cells. The current study was conducted to determine whether disruption of AA homeostasis influences breast cancer cell proliferation and death. Initial experiments revealed that inhibition of AA remodeling through membrane phospholipids by inhibitors of the enzyme, coenzyme A-independent transacylase (CoA-IT), attenuates the proliferation of the estrogen receptor-negative, MDA-MB-231, and estrogen receptor-positive, MCF-7 breast cancer cell lines. This growth inhibition was accompanied by a marked accumulation of AA in both free fatty acid and triglyceride forms, a marker of intracellular AA stress within mammalian cells. Cell cycle synchronization experiments revealed that the CoA-IT inhibitor, SB-98625, blocked MDA-MB-231 cell replication in early to mid G1 phase. Time-lapse video microscopy, used to observe the changes in cell morphology associated with apoptosis, indicated that SB-98625 treatment induced early rounding and occasional blebbing but not late apoptotic events, blistering, and lysis. The cyclooxygenase inhibitors, NS-398 and indomethacin, were found to be less potent blockers of cell proliferation and poor inducers of cellular AA accumulation than CoA-IT inhibitors in these breast cancer cell lines. Finally, AA provided exogenously blocked the proliferation of MCF-7 cells, and this effect could be attenuated in MCF-7 cells overexpressing the glutathione peroxidase gene, GSHPx-1. Taken together, these experiments suggest that disruption of AA remodeling in a manner that increases intracellular AA may represent a novel therapeutic strategy to reduce cancer cell proliferation and that an oxidized AA metabolite is likely to mediate this effect.
Assuntos
Aciltransferases/antagonistas & inibidores , Apoptose , Ácido Araquidônico/metabolismo , Neoplasias da Mama/tratamento farmacológico , Inibidores de Ciclo-Oxigenase/farmacologia , Aciltransferases/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ácidos Graxos não Esterificados/farmacologia , Humanos , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
OBJECTIVES: There is growing evidence of ongoing, in utero neurological damage in fetuses with myelomeningocele (MMC). Phospholipase A2 (PLA2) has known neurotoxic properties and is predominantly present in its secretory isoform (sPLA2) in meconium, the passage of which is increased in MMC fetuses. The objective of this study was to determine if amniotic fluid (AF) levels of PLA2 are elevated in a rat model of MMC. METHODS: Timed pregnant Sprague-Dawley rats were gavage fed 60 mg/kg/bodyweight retinoic acid (RA) in olive oil at embryonic day 10 (E10). Amniocentesis was performed at multiple gestational time points on MMC fetuses, RA-exposed fetuses without MMC and control fetuses. AF PLA2 levels were analyzed by a fluorescent enzyme activity assay. PLA2 isoforms were determined by measuring activity in the presence of specific inhibitors. RESULTS: There was no difference in AF PLA2 activity between groups on E15. PLA2 activity was significantly increased in MMC fetuses on E17, E19 and E21 (p < 0.001). Secretory PLA2 primarily accounted for the overall greater activity. CONCLUSIONS: PLA2 levels are elevated in the AF of fetal rats with MMC and may contribute to ongoing neural injury. This pathway may be a useful drug target to limit ongoing damage and better preserve neurologic function.
Assuntos
Líquido Amniótico/enzimologia , Doenças Fetais/enzimologia , Meningomielocele/enzimologia , Fosfolipases A2 Secretórias/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Doenças Fetais/induzido quimicamente , Imunofluorescência , Isoenzimas/metabolismo , Meningomielocele/induzido quimicamente , Gravidez , Ratos , Ratos Sprague-Dawley , TretinoínaRESUMO
The current study examined mechanisms that account for the selective release of arachidonic acid (AA) from cells by secretory phospholipase A2 (sPLA2). Initial studies demonstrated that low concentrations of group I and group III PLA2 isotypes and an sPLA2-enriched extract from bone marrow-derived mast cells (BMMC) selectively released AA from mast cells. Much higher concentrations of group II PLA2 were required to release comparable quantities of AA. Group I PLA2 also selectively released AA from another mast cell line (CFTL-15) and a monocytic cell line (THP-1). In contrast, high concentrations of group I PLA2 were required to release fatty acids from a promyelocytic cell line (HL-60) and this release was not selective for AA. Binding studies revealed that cell types (BMMC, CFTL-15 and THP-1) which selectively released AA also had the capacity to specifically bind group I PLA2. However, group II PLA2, which did not selectively release AA from cells, also did not specifically bind to these same cell types. Additional studies revealed that sPLA2 binding to the mast cell receptor was attenuated after stimulation with antigen or ionophore A23187. Reverse transcriptase-polymerase chain reaction analyses indicated the presence of mRNA for the sPLA2 receptor in BMMC, CFTL-15 and THP-1 and the absence of this mRNA in HL-60. Final studies demonstrated that p-aminophenyl-alpha-D-mannopyranoside BSA, a known ligand of the sPLA2 receptor, also selectively released AA from mast cells but not from HL-60 cells. These experiments indicated that receptor occupancy alone (without PLA2 activity) is sufficient to induce the release of AA from mast cells. Together, these data reveal that specific isotypes of sPLA2 have the capacity to selectively release AA from certain cells by their capacity to bind to sPLA2 receptors on the cell surface.
Assuntos
Ácido Araquidônico/metabolismo , Células da Medula Óssea/metabolismo , Isoenzimas/farmacologia , Fosfolipases A/farmacologia , Animais , Células da Medula Óssea/enzimologia , Linhagem Celular , Ácidos Graxos/análise , Fosfolipases A2 do Grupo II , Radioisótopos do Iodo , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos CBA , Fosfolipases A/metabolismo , Fosfolipases A2 , Receptores de Superfície Celular/metabolismoRESUMO
This study has examined the metabolism of arachidonic acid in the mouse bone marrow-derived mast cell (BMMC) during immunologic and nonimmunologic activation. The predominant pools of endogenous arachidonate in the mast cells were found in ethanolamine (46%), choline (39%) and inositol (14%) containing glycerolipids. Initial studies established conditions where equilibrium labelling of these major phospholipids in the BMMC could be reached. Upon challenge, arachidonate was lost from all major phospholipid classes (phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylinositol). There was a small but significant increase in the amount of label associated with phosphatidic acid during cell activation. Arachidonate was distributed among 1-acyl, 1-alkyl and 1-alk-1-enyl-linked subclasses of PC and PE. The rank order of loss of labelled arachidonate from the major PE and PC subclasses during antigen and ionophore activation was 1-alk-enyl-2-arachidonoyl-GPE greater than 1-acyl-2-arachidonoyl-GPC greater than 1-acyl-2-arachidonoyl-GPE greater than 1-alkyl-2-arachidonoyl-GPC. Labelled products released into the supernatant fluids and free arachidonic acid within the cell accounted for the bulk of arachidonate lost from phospholipids. Labelled products in the supernatant fluids were composed of LTB4, LTC4, PGD2 and free arachidonic acid. BMMC phospholipids were also labelled for 24 hr with [3H]choline, [3H]myoinositol or [14H]ethanolamine and labelled 2-lyso phospholipids were measured after cell activation. Radioactivity in lysophospholipids from PC, PE and PI increased significantly between 30 s and 2 min after antigen activation and then declined. Taken together, these studies suggest that arachidonate is mobilized predominantly from PE and in particular 1-alk-1-enyl-2-arachidonoyl-GPE by the direct removal of arachidonate from the sn-2 position of the molecule. Most of this arachidonate is then released from cells as eicosanoids or free fatty acid.
Assuntos
Ácidos Araquidônicos/metabolismo , Células da Medula Óssea , Mastócitos/metabolismo , Animais , Antígenos/imunologia , Calcimicina/farmacologia , Células Cultivadas , Cromatografia Gasosa-Espectrometria de Massas , Cinética , Mastócitos/imunologia , Camundongos , Fosfolipídeos/metabolismoRESUMO
BACKGROUND: The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) has become a potential target for the prevention and treatment of breast cancer. However, recent in vitro and in vivo studies have raised the question of whether activation of PPARgamma leads to the promotion or reduction of tumor formation. Studies using several cancer cell lines, animal models, and a variety of PPARgamma agonists have shown discordant results, including changes in cellular proliferation, differentiation, and apoptosis of cancer cells and tumors. METHODS: We studied the effects of low-, moderate-, and high-dose treatment of the PPARgamma ligands 15-deoxy-delta1214 prostaglandin J2 (15dPGJ2) and troglitazone (TGZ) on parameters of cell growth, differentiation, and apoptosis in the epithelial breast cancer cell line MDA-MB-231. RESULTS: The biologic effects of these compounds depend largely on ligand concentration and the degree of PPARgamma activation. For example, low concentrations of 15dPGJ2 (<2.5 microM) and TGZ (<5 microM) increased cellular proliferation, but concentrations of 15dPGJ2 > or = 10 microM and of TGZ at 100 microM blocked cell growth. TGZ (100 microM) slowed cell cycle progression, and 15dPGJ2 (10 microM) caused an S-phase arrest in the cell cycle and induced morphological characteristics consistent with apoptosis. Expression of CD36, a marker of differentiation in these cells, was induced by 2.5 microM 15dPGJ2 or 5 to 100 microM TGZ. However, higher concentrations of 15dPGJ2 did not alter CD36 expression. Transcriptional activation studies demonstrated that 15dPGJ2 is a more potent PPARgamma ligand than TGZ. Regardless of the ligand used, though, low transcriptional activation correlated with an increased cellular proliferation, whereas higher levels of activation correlated with cell cycle arrest and apoptosis. CONCLUSIONS: PPARgamma activation induces several important and seemingly opposite changes in neoplastic cells, depending on the magnitude of PPARgamma activation. These data may explain, at least in part, some of the discordant results previously reported.
Assuntos
Neoplasias da Mama/patologia , Cromanos/farmacologia , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Tiazóis/farmacologia , Tiazolidinedionas , Fatores de Transcrição/fisiologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Feminino , Humanos , Ativação Transcricional/efeitos dos fármacos , Troglitazona , Células Tumorais CultivadasAssuntos
Ácido Araquidônico/metabolismo , Inflamação/metabolismo , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Acetil-CoA Hidrolase/metabolismo , Acilação , Aciltransferases/metabolismo , Animais , Células Cultivadas , Coenzima A Ligases/metabolismo , Eicosanoides/biossíntese , Fosfolipídeos/metabolismoAssuntos
Neoplasias/metabolismo , Prostaglandina D2/análogos & derivados , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Humanos , Neoplasias/patologia , Prostaglandina D2/fisiologiaAssuntos
Mastócitos/enzimologia , Fosfolipases A/isolamento & purificação , RNA Mensageiro/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Primers do DNA , Fosfolipases A2 do Grupo II , Humanos , Fosfolipases A/análise , Fosfolipases A/genética , Fosfolipases A/metabolismo , RoedoresRESUMO
Our aim was to develop a liquid chromatography and electrospray ionization tandem mass spectrometry (LCMS2) method to measure free amino acid (FAA) and dipeptide (DP) concentrations in biological fluids. We synthesized chloroformate derivatives of FAA and DP, identified the major precursor ions and used LCMS2 to obtain the most intense product ions. Using serial dilutions of unlabeled and labeled standards ([2H3]-L-Dopa, homoarginine, homophenylalanine, [15N]-Glutamine and [2H3]-methionine), we observed linear relationships in MS response that we used to calculate the amounts of FAA and DP in biological samples. This method is sensitive with a limit of detection (LOD) for most of the FAAs and DPs tested in the 0.05-1 pmol range and is linear over 3-5 orders of magnitude when many metabolites were measured simultaneously. Reproducibility and between run or daily variations were <10% for most FAAs and DPs. We applied this method to human samples and quantitatively measured 21 FAAs and 2 DPs in 200 microl CSF, 31 FAAs and 6 DPs in 100 microl plasma, and 23 FAAs and 5 DPs in 200 microl urine. These data demonstrate the potential for using LCMS2 to discover changes in FAA and DP metabolic pathways that occur during disease pathogenesis.
Assuntos
Aminoácidos/sangue , Aminoácidos/líquido cefalorraquidiano , Aminoácidos/urina , Cromatografia Líquida/métodos , Dipeptídeos/sangue , Dipeptídeos/líquido cefalorraquidiano , Dipeptídeos/urina , Espectrometria de Massas por Ionização por Electrospray/métodos , Líquidos Corporais/metabolismo , Líquido Cefalorraquidiano/metabolismo , Humanos , Íons , Levodopa/farmacologia , Espectrometria de Massas/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Our aim was to determine changes in free amino acid (FAA) and dipeptide (DP) concentrations in probable Alzheimer's disease (pAD) subjects compared with control (CT) subjects using liquid chromatography and electrospray ionization tandem mass spectrometry (LCMS2). We recruited gender- and age-matched study participants based on neurological and neuropsychological assessments. We measured FAAs and DPs in cerebrospinal fluid (CSF), plasma and urine using LCMS2 with selected reaction monitoring (SRM). Imidazole-containing FAAs (histidine, methyl-histidine), catecholamines (L-DOPA and dopamine), citrulline, ornithine, glycine and antioxidant DPs (carnosine and anserine) accounted for the major changes between CT and pAD. Carnosine levels were significantly lower in pAD (328.4 +/- 91.31 nmol/dl) than in CT plasma (654.23 +/- 100.61 nmol/dl). In contrast, L-DOPA levels were higher in pAD (1400.84 +/- 253.68) than CT (513.10 +/- 121.61 nmol/dl) plasma. These data underscore the importance of FAA and DP metabolism in the pathogenesis of AD. Since our data show changes in antioxidants, neurotransmitters and their precursors, or FAA associated with urea metabolism in pAD compared with CT, we propose that manipulation of these metabolic pathways may be important in preventing AD progression.
Assuntos
Doença de Alzheimer/sangue , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/urina , Aminoácidos/sangue , Aminoácidos/líquido cefalorraquidiano , Aminoácidos/urina , Dipeptídeos/sangue , Dipeptídeos/líquido cefalorraquidiano , Dipeptídeos/urina , Idoso , Antioxidantes/química , Carnosina/análise , Cromatografia Líquida , Progressão da Doença , Feminino , Histidina/química , Humanos , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The objective of the present study was to better understand the remodeling of arachidonic acid (AA) in phospholipids of the mouse bone marrow-derived mast cell (BMMC) during Ag and ionophore A23187 activation. Initial studies were designed to understand the movement of AA in phospholipid classes under resting conditions. BMMC pulse labeled with AA incorporated greater than 95% of the label into the major phospholipid classes. Phosphatidylcholine (PC) subclasses, 1-acyl-2-arachidonoyl-(sn-glycero-3-phosphocholine (GPC)) in particular, initially accounted for most of the label incorporated into the cells with phosphatidylinositol/phosphatidylserine (PI/PS) and phosphatidylethanolamine (PE) subclasses containing much smaller quantities. Prolonged incubation of labeled BMMC resulted in a decrease in the radioactivity in PC with a concomitant increase in PE such that 1-alk-1-enyl-2-arachidonoyl-(sn-glycero-3-phosphoethanolamine (GPE)) became the single largest labeled AA pool by 24 h. Further experiments indicated that 24 h was the time required to reach isotopic equilibrium among AA-containing phospholipids of the BMMC. In the next series of experiments, BMMC phospholipids were labeled to different specific activities by either labeling the cells for 0.5 h or for 24 h followed by stimulation. Under isotopic equilibrium conditions (24 h), stimulation resulted in AA release from PE greater than PC much greater than PI/PS with 1-alk-1-enyl-2-arachidonoyl-GPE providing the bulk of AA released from the BMMC. By contrast, cells labeled for 0.5 h released AA from PC much greater than PI/PS, with 1-acyl-2-arachidonoyl-GPC accounting for most of the AA released from BMMC phospholipids. Label associated with PE subclasses under nonequilibrium conditions remained unchanged or slightly increased throughout a 10-min stimulation period. Finally, BMMC were double labeled with [14C]-AA for 24 h and then with [3H]-AA for 0.5 h. Cell stimulation resulted in a decrease in the [3H]/[14C] ratio in PC and PI and an increase in the ratio in PE. The decrease in [3H]/[14C] ratio in PC was mainly in 1-acyl-2-arachidonoyl-GPC, whereas the increase in PE subclasses was primarily in 1-alk-1-enyl-2-arachidonoyl-GPE. The [3H]/[14C] ratio in cellular neutral lipids and in supernatant fluid products were at values between PC and PE subclasses. Taken together, these data suggest that during Ag activation, the release of free arachidonic acid is from predominantly PE subclasses. Concomitant with the release of AA, there is a rapid remodeling of AA from PC subclasses into PE subclasses (1-alk-1-enyl-2-acyl-GPE).
Assuntos
Ácido Araquidônico/metabolismo , Mastócitos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Animais , Antígenos/administração & dosagem , Calcimicina/farmacologia , Divisão Celular , Mastócitos/citologia , Lipídeos de Membrana/metabolismo , Camundongos , Fatores de TempoRESUMO
Immunologic activation of mast cells leads to the mobilization of arachidonic acid (AA) from membrane phospholipids and the subsequent conversion of this AA to bioactive products. The objective of our study was to determine if segregated pools of AA-containing phospholipids within mast cells serve as independent sources of AA. Initial studies indicated that the appearance of free AA occurred rapidly (maximal level formed within 1 min) within supernatant fluids of Ag-stimulated mast cells and was kinetically different from the formation of leukotriene (LT) B4 or LTC4. To examine whether free AA and leukotrienes were mobilized from different sources, AA-containing phospholipids of mast cells were labeled with [14C] and [3H] AA such that all major subclasses (1-acyl-1-alkyl-1-alk-1'-enyl) of phospholipids contained different ratios of [3H] to [14C] (sp. act. ratios (SAR)). Mast cells were then stimulated with Ag and the SAR of cellular AA, extracellular AA and extracellular LTC4, LTB4, 6-trans LTB4, were determined. The SAR were uniform in all LT and mimicked the ratio found in cellular AA. By contrast, the SAR of AA released into supernatant fluids was twofold lower than that of LT. This indicated that AA released as free fatty acid clearly was derived from a different lipid pool than AA that formed LT. Although it was apparent that the pools which gave rise to AA and LT were different, defining phospholipid(s) that constitute these distinct pools proved more difficult. The SAR of LT suggested that their cellular precursor AA could have been derived from several phospholipid subclasses; however, the SAR in phosphatidylcholine and phosphatidylinositol most closely matched the LT. The SAR of AA in supernatant fluids implied that it was derived, in part, from phosphatidylethanolamine subclasses. Taken together, these data suggest that there are at least two different pools of AA that are mobilized in mast cells during Ag activation.
Assuntos
Antígenos/imunologia , Ácido Araquidônico/metabolismo , Leucotrienos/biossíntese , Mastócitos/imunologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos CBA , Fosfolipídeos/metabolismoRESUMO
Recent studies have demonstrated that inflammatory cells can be divided into two groups depending on the type of 2-acetylated phospholipids [1-radyl-2-acetyl-sn-glycero-3-phosphocholine (GPC)] they produce: those that produce predominantly platelet-activating factor (PAF), and those that produce predominantly its 1-acyl analogue (1-acyl-2-acetyl-GPC; AAGPC) [Triggiani, Schleimer, Warner & Chilton (1991) J. Immunol. 147, 660-666]. The present study has examined the factors that regulate the production of these two molecules in mouse bone marrow-derived mast cells (BMMC). Initial experiments indicated that PAF and AAGPC were catabolized by BMMC in a differential manner via two pathways: the first, exclusive for AAGPC, involved a 1-acyl hydrolase that removed the long chain at the sn-1 position of the molecule, and the second, common to AAGPC and PAF, involved acetylhydrolase that removed the acetate at the sn-2 position of the two molecules. Experiments were next designed to identify conditions where the differential catabolism of AAGPC and PAF could be eliminated in order to uncover other factors that regulate the proportions of AAGPC and PAF produced. Phenylmethanesulphonyl fluoride (PMSF) completely blocked the 1-acylhydrolase activity while having little or no effect on the acetyl hydrolase activity, thereby eliminating the influence of the catabolic pathway unique to AAGPC. Moreover, PMSF did not alter the release of arachidonic acid from phospholipid subclasses. PMSF-treated BMMC produced larger quantities of AAGPC than of PAF. The AAGPC/PAF ratio detected in PMSF-treated BMMC was very similar to the ratio of arachidonate contained in and released from 1-acyl-/1-alkyl-linked phosphatidylcholine (PC). BMMC supplemented with arachidonic acid in culture for 3 days increased their total arachidonic acid content in PC as well as the ratio of 1-acyl-2-arachidonoyl-GPC to 1-alkyl-2-arachidonoyl-GPC. These changes resulted in parallel and significant increases in both the total amount of 1-radyl-2-acetyl-GPC and the AAGPC/PAF ration in BMMC. These data indicate that the AAGPC/PAF ratio produced by inflammatory cells is regulated by at least two factors: (1) differential catabolism of these two molecules, and (2) the distribution of arachidonate in 1-acyl- and 1-alkyl-2-arachidonyl-GPC. These observations support the concept of a common pathway for AAGPC and PAF biosynthesis in which the two precursor molecules are 1-acyl-2-arachidonoyl-GPC and 1-alkyl-2-arachidonoyl-GPC, respectively.
Assuntos
Mastócitos/metabolismo , Fator de Ativação de Plaquetas/análogos & derivados , Fator de Ativação de Plaquetas/biossíntese , Animais , Ácido Araquidônico/metabolismo , Calcimicina/farmacologia , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos CBA , Fluoreto de Fenilmetilsulfonil/farmacologia , Fósforo/metabolismoRESUMO
Phospholipase A2 (PLA2) is a pivotal enzyme involved in the synthesis of the potent lipid inflammatory mediators platelet activating factor (PAF) and the eicosanoids. This study characterizes a PLA2 recovered in the bronchoalveolar lavage fluid (BALF) of healthy adult human subjects. Human BALF PLA2 exhibited characteristics of secretory PLA2s that include an activity that is acid stable, sensitive to reducing agents, and optimally requires millimolar calcium. BALF PLA2 showed marked selectivity for phosphatidylcholine containing arachidonic acid (AA) over linoleic or palmitic acids. Size exclusion chromatography showed the BALF PLA2 protein to be approximately 14 kDa in mass, consistent with it being a secretory form of PLA2. The biological significance of BALF PLA2 was tested by applying BALF concentrates to cultures of the human bronchial epithelial cell line BEAS 2B. Cultures of BEAS 2B cells treated with BALF concentrates released increased amounts of AA and produced higher levels of PAF. These data show that the lining fluid of the human respiratory tract normally contains a secretory PLA2, which may be involved in the formation of lipid inflammatory mediators in normal and pathophysiologic states in the lung.
Assuntos
Líquido da Lavagem Broncoalveolar/química , Pulmão/enzimologia , Fosfolipases A/metabolismo , Adolescente , Adulto , Ácido Araquidônico/análise , Ácido Araquidônico/metabolismo , Cálcio/análise , Linhagem Celular/enzimologia , Cromatografia Líquida de Alta Pressão , Humanos , Pulmão/metabolismo , Fosfolipases A/análise , Fosfolipases A2 , Fator de Ativação de Plaquetas/análise , Fator de Ativação de Plaquetas/metabolismoRESUMO
Our previous studies demonstrated that inhibitors of arachidonate-phospholipid remodeling [i.e. the enzyme CoA-independent transacylase (CoA-IT)] decrease cell proliferation and induce apoptosis in neoplastic cells. The goal of the current study was to elucidate the molecular events associated with arachidonate-phospholipid remodeling that influence cell proliferation and survival. Initial experiments revealed the essential nature of cellular arachidonate to the signaling process by demonstrating that HL-60 cells depleted of arachidonate were more resistant to apoptosis induced by CoA-IT inhibition. In cells treated with CoA-IT inhibitors a marked increase in free arachidonic acid and AA-containing triglycerides were measured. TG enrichment was likely due to acylation of arachidonic acid into diglycerides and triglycerides via de novo glycerolipid biosynthesis. To determine the potential of free fatty acids to affect cell proliferation, HL-60 cells were incubated with varying concentrations of free fatty acids; exogenously provided 20-carbon polyunsaturated fatty acids caused a dose-dependent inhibition of cell proliferation, whereas oleic acid was without effect. Blocking 5-lipoxygenase or cyclooxygenases had no effect on the inhibition of cell proliferation induced by arachidonic acid or CoA-IT inhibitors. An increase in cell-associated ceramides (mainly in the 16:0-ceramide fraction) was measured in cells exposed to free arachidonic acid or to CoA-IT inhibitors. This study, in conjunction with other recent studies, suggests that perturbations in the control of cellular arachidonic acid levels affect cell proliferation and survival.