Urine dicarboxylic acids change in pre-symptomatic Alzheimer's disease and reflect loss of energy capacity and hippocampal volume.

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.

Amniotic fluid levels of phospholipase A2 in fetal rats with retinoic acid induced myelomeningocele: the potential "second hit" in neurologic damage.

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.

Free amino acid and dipeptide changes in the body fluids from Alzheimer's disease subjects.

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.

Quantification of free amino acids and dipeptides using isotope dilution liquid chromatography and electrospray ionization tandem mass spectrometry.

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.

Enhancement of mast cell survival: a novel function of some secretory phospholipase A(2) isotypes.

This study tested the hypothesis that certain secretory phospholipase A(2) (sPLA(2)) isotypes act in a cytokine-like fashion through cell surface receptors to influence mast cell survival. Initial experiments revealed that sPLA(2) activity and sPLA(2) receptor expression are increased, and mast cells lost their capacity to maintain membrane asymmetry upon cytokine depletion. Groups IB and III, but not group IIA PLA(2), prevented the loss of membrane asymmetry. Similarly, group IB prevented nucleosomal DNA fragmentation in mast cells. Providing putative products of sPLA(2) hydrolysis to cytokine-depleted mast cells did not influence survival. Furthermore, catalytic inactivation of sPLA(2) did not alter its capacity to prevent apoptosis. Inhibition of protein synthesis using cycloheximide or actinomycin reversed the antiapoptotic effect of sPLA(2). Additionally, both wild-type and catalytically inactive group IB PLA(2) induced IL-3 synthesis in mast cells. However, adding IL-3-neutralizing Ab did not change Annexin V(FITC) binding and only partially inhibited thymidine incorporation in sPLA(2)-supplemented mast cells. In contrast, IL-3-neutralizing Ab inhibited both Annexin V(FITC) binding and thymidine incorporation in mast cells maintained with IL-3. sPLA(2) enhanced phosphoinositide 3'-kinase activity, and a specific inhibitor of phosphoinositide 3'-kinase reversed the antiapoptotic effects of sPLA(2). Likewise, sPLA(2) increased the degradation of I-kappaBalpha, and specific inhibitors of nuclear factor kappa activation (NF-kappaB) reversed the antiapoptotic effects of sPLA(2). Together, these experiments reveal that certain isotypes of sPLA(2) enhance the survival of mast cells in a cytokine-like fashion by activating antiapoptotic signaling pathways independent of IL-3 and probably via sPLA(2) receptors rather than sPLA(2) catalytic products.

Magnitude of peroxisome proliferator-activated receptor-gamma activation is associated with important and seemingly opposite biological responses in breast cancer cells.

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.

A decrease in remodeling accounts for the accumulation of arachidonic acid in murine mast cells undergoing apoptosis.

The goal of this study was to examine arachidonic acid (AA) metabolism by murine bone marrow-derived mast cells (BMMC) during apoptosis induced by cytokine depletion. BMMC deprived of cytokines for 12-48 h displayed apoptotic characteristics. During apoptosis, levels of AA, but not other unsaturated fatty acids, correlated with the percentage of apoptotic cells. A decrease in both cytosolic phospholipase A(2) expression and activity indicated that cytosolic phospholipase A(2) did not account for AA mobilization during apoptosis. Free AA accumulation is also unlikely to be due to decreases in 5-lipoxygenase and/or cyclooxygenase activities, since BMMC undergoing apoptosis produced similar amounts of leukotriene B(4) and significantly greater amounts of PGD(2) than control cells. Arachidonoyl-CoA synthetase and CoA-dependent transferase activities responsible for incorporating AA into phospholipids were not altered during apoptosis. However, there was an increase in arachidonate in phosphatidylcholine (PC) and neutral lipids concomitant with a 40.7 +/- 8.1% decrease in arachidonate content in phosphatidylethanolamine (PE), suggesting a diminished capacity of mast cells to remodel arachidonate from PC to PE pools. Further evidence of a decrease in AA remodeling was shown by a significant decrease in microsomal CoA-independent transacylase activity. Levels of lyso-PC and lyso-PE were not altered in cells undergoing apoptosis, suggesting that the accumulation of lysophospholipids did not account for the decrease in CoA-independent transacylase activity or the induction of apoptosis. Together, these data suggest that the mole quantities of free AA closely correlated with apoptosis and that the accumulation of AA in BMMC during apoptosis was mediated by a decreased capacity of these cells to remodel AA from PC to PE.

Secretory phospholipase A2 receptor-mediated activation of cytosolic phospholipase A2 in murine bone marrow-derived mast cells.

The current study examined the signal transduction steps involved in the selective release of arachidonic acid (AA) induced by the addition of secretory phospholipase A2 (sPLA2) isotypes to bone marrow-derived mast cells (BMMC). Overexpression of sPLA2 receptors caused a marked increase in AA and PGD2 release after stimulation of BMMC, implicating sPLA2 receptors in this process. The hypothesis that the release of AA by sPLA2 involved activation of cytosolic PLA2 (cPLA2) was next tested. Addition of group IB PLA2 to BMMC caused a transient increase in cPLA2 activity and translocation of this activity to membrane fractions. Western analyses revealed that these changes in cPLA2 were accompanied by a time-dependent gel shift of cPLA2 induced by phosphorylation of cPLA2 at various sites. A noncatalytic ligand of the sPLA2 receptor, p-amino-phenyl-alpha-D-mannopyranoside BSA, also induced an increase in cPLA2 activity in BMMC. sPLA2 receptor ligands induced the phosphorylation of p44/p42 mitogen-activated protein kinase. Additionally, an inhibitor of p44/p42 mitogen-activated protein kinase (PD98059) significantly inhibited sPLA2-induced cPLA2 activation and AA release. sPLA2 receptor ligands also increased Ras activation while an inhibitor of tyrosine phosphorylation (herbimycin) inhibited the increase in cPLA2 activation and AA release. Addition of partially purified sPLA2 from BMMC enhanced cPLA2 activity and AA release. Similarly, overexpression of mouse groups IIA or V PLA2 in BMMC induced an increase in AA release. These data suggest that sPLA2 mediate the selective release of AA by binding to cell surface receptors and then inducing signal transduction events that lead to cPLA2 activation.

Addition of eicosapentaenoic acid to gamma-linolenic acid-supplemented diets prevents serum arachidonic acid accumulation in humans.

Previous studies reveal that supplementation of human diets with gamma-linolenic acid (GLA) reduces the generation of lipid mediators of inflammation and attenuates clinical symptoms of chronic inflammatory disorders such as rheumatoid arthritis. However, we have shown that supplementation with this same fatty acid also causes a marked increase in serum arachidonate (AA) levels, a potentially harmful side effect. The objective of this study was to design a supplementation strategy that maintained the capacity of GLA to reduce lipid mediators without causing elevations in serum AA levels. Initial in vitro studies utilizing HEP-G2 liver cells revealed that addition of eicosapentaenoic acid (EPA) blocked Delta-5-desaturase activity, the terminal enzymatic step in AA synthesis. To test the in vivo effects of a GLA and EPA combination in humans, adult volunteers consuming controlled diets supplemented these diets with 3.0 g/d of GLA and EPA. This supplementation strategy significantly increased serum levels of EPA, but did not increase AA levels. EPA and the elongation product of GLA, dihomo-gamma-linolenic acid (DGLA) levels in neutrophil glycerolipids increased significantly during the 3-wk supplementation period. Neutrophils isolated from volunteers fed diets supplemented with GLA and EPA released similar quantities of AA, but synthesized significantly lower quantities of leukotrienes compared with their neutrophils before supplementation. This study revealed that a GLA and EPA supplement combination may be utilized to reduce the synthesis of proinflammatory AA metabolites, and importantly, not induce potentially harmful increases in serum AA levels.

Influence of J series prostaglandins on apoptosis and tumorigenesis of breast cancer cells.

This study was undertaken to investigate the influence of the peroxisome proliferator-activated receptor gamma (PPARgamma) agonists on the proliferation, apoptosis and tumorigenesis of breast cancer cells. PPARgamma investigation has been largely restricted to adipose tissue, where it plays a key role in differentiation, but recent data reveal that PPARgamma is expressed in several transformed cells. However, the function of PPARgamma activation in neoplastic cells is unclear. Activation of PPARgamma with the known prostanoid agonist 15-deoxy-Delta12,14-prostaglandin J(2) (15dPGJ(2)) or the thiazolidinedione (TZD) agonist troglitazone (TGZ) attenuated cellular proliferation of the estrogen receptor-negative breast cancer cell line MDA-MB-231, as well as the estrogen receptor-positive breast cancer cell line MCF-7. This was marked by a decrease in total cell number and by an inhibition of cell cycle progression. Addition of 15dPGJ(2) was not associated with an increase in cellular differentiation, as has been seen in other neoplastic cells, but rather induction of cellular events associated with programmed cell death, apoptosis. Video time-lapse microscopy revealed that 15dPGJ(2) induced morphological changes associated with apoptosis, including cellular rounding, blebbing, the production of echinoid spikes, blistering and cell lysis. In contrast, TGZ caused only a modest induction of apoptosis. These results were verified by histochemistry using the specific DNA stain DAPI to observe nuclear condensation, a marker of apoptosis. Finally, a brief exposure of MDA-MB-231 cells to 15dPGJ(2) initiated an irreversible apoptotic pathway that inhibited the growth of tumors in a nude mouse model. These findings illustrate that induction of apoptosis may be the primary biological response resulting from PPARgamma activation in some breast cancer cells and further suggests a potential role for PPARgamma ligands for the treatment of breast cancer.

Role of arachidonyl triglycerides within lipid bodies in eicosanoid formation by human polymorphonuclear cells.

Increasing evidence suggests that the subcellular and glycerolipid localization of esterified arachidonic acid (AA) is a key factor in regulating its availability to lipases. The goal of the current study was to determine the potential of AA stored in triglycerides (TG) to serve as a substrate for lipases and 5-lipoxygenase during neutrophil (polymorphonuclear leukocytes, PMN) activation. PMN containing high concentrations of AA in TG were generated by culturing PMN in vitro with high concentrations of exogenous AA (eAA) for 12 h. Cellular AA increased 2- and 4-fold in PMNs incubated with 5 and 20 microM AA, respectively, and this increase was almost exclusively observed in neutral lipids (NL). Further analysis revealed that 88% of the AA in the NL fraction was associated with TG. Subsequent experiments were designed to determine whether this AA in TG could be mobilized and metabolized to eicosanoids during cell activation. TG pools of AA were increased as previously described and then PMN were stimulated with ionophore, A23187. In contrast to the 43-fold increase in TG AA after eAA loading (20 microM), free AA increased by only 1.9-fold after cell stimulation. Similarly, leukotriene (LT)B(4) production increased only 2-fold after loading TG with large quantities of AA. The magnitude of increase in free AA released and in LTB(4) formation was similar to the magnitude of increase in AA mass in phospholipase (PL), suggesting that PL, and not TG, served as the source of released AA and subsequent product generation. To confirm that AA in TG did not serve as a source for eicosanoid production, cellular pools of AA were differentially labeled with [(14)C]AA and [(3)H]AA, and the [(3)H]AA-to-[(14)C]AA ratio of LTB(4) and 20-hydroxyl LTB(4) produced during cell stimulation was measured. The [(3)H]AA/[(14)C]AA ratios of LTs were markedly different from the ratios in TG, thus providing further evidence that AA pools in TG are not a major source of AA for LT generation.

Perturbations in the control of cellular arachidonic acid levels block cell growth and induce apoptosis in HL-60 cells.

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.

Influence of coenzyme A-independent transacylase and cyclooxygenase inhibitors on the proliferation of breast cancer cells.

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.

Mechanisms that account for the selective release of arachidonic acid from intact cells by secretory phospholipase A2.

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.

Dietary supplementation with gamma-linolenic acid alters fatty acid content and eicosanoid production in healthy humans.

To understand the in vivo metabolism of dietary gamma-linolenic acid (GLA), we supplemented the diets of 29 volunteers with GLA in doses of 1.5-6.0 g/d. Twenty-four subjects ate controlled eucaloric diets consisting of 25% fat; the remaining subjects maintained their typical Western diets. GLA and dihomo-gamma-linolenic acid (DGLA) increased in serum lipids of subjects supplemented with 3.0 and 6.0 g/d; serum arachidonic acid increased in all subjects. GLA supplementation with 3.0 and 6.0 g/d also resulted in an enrichment of DGLA in neutrophil phospholipids but no change in GLA or AA levels. Before supplementation, DGLA was associated primarily with phosphatidylethanolamine (PE) of neutrophil glycerolipids, and DGLA increased significantly in PE and neutral lipids after GLA supplementation. Extending the supplementation to 12 wk did not consistently change the magnitude of increase in either serum or neutrophil lipids in subjects receiving 3.0 g/d. After GLA supplementation, A23187-stimulated neutrophils released significantly more DGLA, but AA release did not change. Neutrophils obtained from subjects after 3 wk of supplementation with 3.0 g/d GLA synthesized less leukotriene B4 (P < 0.05) and platelet-activating factor. Together, these data reveal that DGLA, the elongase product of GLA, but not AA accumulates in neutrophil glycerolipids after GLA supplementation. The increase in DGLA relative to AA within inflammatory cells such as the neutrophil may attenuate the biosynthesis of AA metabolites and may represent a mechanism by which dietary GLA exerts an anti-inflammatory effect.

Relationship between arachidonate--phospholipid remodeling and apoptosis.

Our previous studies reveal that three structurally distinct inhibitors of the enzyme CoA-independent transacylase, including the antiproliferative alkyllysophospholipid ET-18-O-CH3, induce programmed cell death (apoptosis) in the promyelocytic cell line HL-60. The objective of the current study was to better elucidate the mechanism responsible for apoptosis. CoA-IT is an enzyme believed to be responsible for the remodeling of long chain polyunsaturated fatty acids like arachidonate between the phospholipids of mammalian cells. The chronic (24-48 h) treatment of HL-60 cells with all three CoA-IT inhibitors resulted in the inhibition of the remodeling of labeled arachidonate from choline- into ethanolamine-containing phospholipid molecular species. GC-MS analysis of the fatty acids in phospholipids revealed that CoA-IT inhibitor treatment induced a marked loss of arachidonate-containing phosphatidylethanolamine and an increase in arachidonate-containing phosphatidylcholine. This redistribution was specific to arachidonate since the mass distribution of linoleic acid in glycerolipids was not affected. In spite of the dramatic redistribution of arachidonate, the total cellular arachidonate content was not altered nor was the relative distribution of total phospholipid classes. The increase of arachidonate in phosphatidylcholine was specifically due to an increase in 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine species, whereas the loss of arachidonate in PE was from both 1-acyl- and 1-alk-1-enyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine species. The incubation of cells with exogenous arachidonic acid or ethanolamine did not reverse the inhibition of proliferation induced by CoA-IT inhibitor treatment. Incubation with CoA-IT inhibitors also induced the characteristic cytoplasmic and nuclear changes associated with apoptosis as assessed by transmission electron microscopy and DNA fragmentation as determined by flow cytometry. Taken together, these data show that apoptosis in HL-60 cells, induced by blocking arachidonate-phospholipid remodeling, is correlated with a redistribution of arachidonate in membrane phospholipids and suggest that such alterations represent a signal which controls the capacity of cells to proliferate.

Alterations in arachidonic acid metabolism in mouse mast cells induced to undergo maturation in vitro in response to stem cell factor.

We studied arachidonic acid (AA) metabolism during the maturation of bone marrow-derived cultured mast cells (BMCMCs) into mast cells with phenotypic characteristics, which were more similar to those of connective tissue-type mast cells. BMCMCs were maintained in medium containing 100 ng/ml recombinant rat stem cell factor (SCF) for 1 to 6 weeks. After 3 to 4 weeks in SCF, BMCMCs acquired many phenotypic characteristics of maturation, including enlarged size, numerous electron-dense cytoplasmic granules, and a 50-fold elevation in histamine content. Maintenance in SCF for 6 weeks did not significantly alter the amounts or species of eicosanoids that were produced by BMCMCs stimulated with calcium ionophore A23187. However, SCF-treated mast cells released 2.6 +/- 0.13 times more free AA and accumulated 6.4 +/- 1.0 times higher levels of intracellular free AA than did immature BMCMCs not exposed to SCF. There was no increase in the mobilization of other fatty acids (e.g., linoleic or oleic acid), indicating specificity for AA. Moreover, there were no differences between the 5-lipoxygenase activities of SCF-treated or untreated cells, as assayed in cell homogenates prepared by nitrogen cavitation. Although the total AA content in SCF-treated cells was significantly elevated, the distribution of AA in phospholipid and neutral lipid classes was not altered by SCF treatment. Total phospholipase (PL)A2 activity increased 85% +/- 11.5% in SCF-treated cells. In homogenates of immature BMCMCs, 51.0% +/- 13.7% of the PLA2 activity was inhibited by 0.5 mmol/L dithiothreitol, whereas the same concentration of dithiothreitol caused only a 2.2% +/- 10.7% reduction in the PLA2 activity in homogenates of SCF-treated BMCMCs (p < or = 0.05, n = 4). These findings suggest that SCF treatment induces a dithiothreitol-resistant PLA2 and that this PLA2 may contribute to the mobilization of AA that is not further metabolized to eicosanoids.