Fatty acid binding proteins stabilize leukotriene A4: competition with arachidonic acid but not other lipoxygenase products

Leukotriene A(4) (LTA(4)) is a chemically reactive conjugated triene epoxide product derived from 5-lipoxygenase oxygenation of arachidonic acid. At physiological pH, this reactive compound has a half-life of less than 3 s at 37 degrees C and approximately 40 s at 4 degrees C. Regardless of this aqueous instability, LTA(4) is an intermediate in the formation of biologically active leukotrienes, which can be formed through either intracellular or transcellular biosynthesis. Previously, epithelial fatty acid binding protein (E-FABP) present in RBL-1 cells was shown to increase the half-life of LTA(4) to approximately 20 min at 4 degrees C. Five FABPs (adipocyte FABP, intestinal FABP, E-FABP, heart/muscle FABP, and liver FABP) have now been examined and also found to increase the half-life of LTA(4) at 4 degrees C to approximately 20 min with protein present. Stabilization of LTA(4) was examined when arachidonic acid was present to compete with LTA(4) for the binding site on E-FABP. Arachidonate has an apparent higher affinity for E-FABP than LTA(4) and was able to completely block stabilization of the latter. When E-FABP is not saturated with arachidonate, FABP can still stabilize LTA(4). Several lipoxygenase products, including 5-hydroxyeicosatetraenoic acid, 5,6-dihydroxyeicosatetraenoic acid, and leukotriene B(4), were found to have no effect on the stability of LTA(4) induced by E-FABP even when present at concentrations 3-fold higher than LTA(4).     

Covalent modification of epithelial fatty acid-binding protein by 4-hydroxynonenal in vitro and in vivo. Evidence for a role in antioxidant biology

4-Hydroxynonenal (4-HNE) is a cytotoxic alpha,beta-unsaturated acyl aldehyde that is naturally produced from lipid peroxidation and cleavage in response to oxidative stress and aging. Such reactive lipids covalently modify cellular target proteins, thereby affecting biological structure and function. Herein we report the identification of the epithelial fatty acid-binding protein (E-FABP) as a molecular target for 4-HNE modification both in vitro and in vivo. 4-HNE covalently modified (t(12) < 60 s) E-FABP in vitro, as revealed by a combination of matrix-assisted laser desorption ionization-time of flight mass spectrometry and immunochemical reactivity using antibodies directed to 4-HNE-protein conjugates. Identification of Cys-120 as the major site of modification was determined through tandem mass spectral sequencing of tryptic peptides, as well as analysis of E-FABP mutants C120A, C127A, and C120A/C127A. The in vitro modification of Cys-120 by 4-HNE was relatively insensitive to pH (6.4-8.4), and temperature (4-37 degrees C) but was markedly potentiated by noncovalently bound fatty acids. 4-HNE-modified E-FABP was more stable than unmodified E-FABP to chemical denaturation by guanidine hydrochloride, as assessed by changes in intrinsic tryptophan fluorescence. Analysis of soluble protein extracts from rat retina with antibodies directed to 4-HNE-protein conjugates revealed immunoreactivity with a 15-kDa protein that was identified by electrospray ionization and matrix-assisted laser desorption ionization-time of flight mass spectrometry as E-FABP. Evaluation of retinal pigment epithelial cell extracts derived from E-FABP null mice by two-dimensional gel electrophoresis using anti-4-HNE antibodies revealed increased modification in the null cells relative to those from wild type cells. These results indicate that E-FABP is a molecular target for 4-HNE modification and the hypothesis that E-FABP functions as an antioxidant protein by scavenging reactive lipids through covalent modification of Cys-120.     

Covalent binding of LTA(4) to nucleosides and nucleotides

Leukotriene A(4) (LTA(4)) is a chemically reactive conjugated triene epoxide that is formed by 5-lipoxygenase and is an intermediate in the formation of the biologically active eicosanoids leukotriene B(4) and leukotriene C(4). The present study was undertaken to determine whether or not LTA(4) could serve as an electrophilic species that nucleosides and nucleotides could attack, ultimately resulting in a covalent adduct. Electrospray ionization mass spectrometry and tandem mass spectrometry were used to study the covalent binding of LTA(4) with uridine, cytidine, adenosine, and guanosine. The reaction with guanosine was found to yield five major and at least six minor adduct species. Reversed phase HPLC and mass spectrometric data suggested that the guanosine attacked LTA(4) either at carbon-12 or carbon-6 with opening the epoxide at carbon-5 to yield a series of adducts characterized by the molecular anion [M-H](-) at m/z 600.3. Reactions of LTA(4) with mixtures of nucleosides and nucleotides revealed that guanine-containing nucleosides were the most reactive toward LTA(4). The facility of the reaction of guanine with LTA(4) raises the possibility that this intermediate of leukotriene biosynthesis formed on or near the cellular nuclear envelope may react with nucleosides and nucleotides present in RNA or DNA.     

Biosynthesis and metabolism of leukotrienes

Leukotrienes are metabolites of arachidonic acid derived from the action of 5-LO (5-lipoxygenase). The immediate product of 5-LO is LTA4 (leukotriene A4), which is enzymatically converted into either LTB4 (leukotriene B4) by LTA4 hydrolase or LTC4 (leukotriene C4) by LTC4 synthase. The regulation of leukotriene production occurs at various levels, including expression of 5-LO, translocation of 5-LO to the perinuclear region and phosphorylation to either enhance or inhibit the activity of 5-LO. Several other proteins, including cPLA2a (cytosolic phospholipase A2a) and FLAP (5-LO-activating protein) also assemble at the perinuclear region before production of LTA4. LTC4 synthase is an integral membrane protein that is present at the nuclear envelope; however, LTA4 hydrolase remains cytosolic. Biologically active LTB4 is metabolized by w-oxidation carried out by specific cytochrome P450s (CYP4F) followed by b-oxidation from the w-carboxy position and after CoA ester formation. Other specific pathways of leukotriene metabolism include the 12-hydroxydehydrogenase/15-oxo-prostaglandin-13-reductase that forms a series of conjugated diene metabolites that have been observed to be excreted into human urine. Metabolism of LTC4 occurs by sequential peptide cleavage reactions involving a g-glutamyl transpeptidase that forms LTD4 (leukotriene D4) and a membrane-bound dipeptidase that converts LTD4 into LTE4 (leukotriene E4) before w-oxidation. These metabolic transformations of the primary leukotrienes are critical for termination of their biological activity, and defects in expression of participating enzymes may be involved in specific genetic disease.     

Leukotriene A4: preparation and enzymatic conversion in a cell-free system to leukotriene B4

Treatment of leukotriene A4 (LTA4) methyl ester with sodium hydroxide in aqueous methanol at 4 degrees C afforded LTA4, the presence of which was inferred from the UV spectrum of the compound, its rate of reaction with water, and the identity of the hydration products obtained. The half-life of LTA4 in water (pH 7.4, room temperature) was increased from 14 to 500 s by 1 mg/ml of bovine serum albumin. This stabilized (chiral) LTA4 was converted to LTB4 by an epoxide hydrolase activity in the 100,000 x g supernatant fraction from sonified rat basophilic leukemia cells. Neither the ester of LTA4 nor the biologically incorrect enantiomer of LTA4 was metabolized to LTB4 under these conditions.     

Expression of E-FABP in PC12 cells increases neurite extension during differentiation: involvement of n-3 and n-6 fatty acids

Epidermal fatty acid-binding protein (E-FABP), a member of the family of FABPs, exhibits a robust expression in neurons during axonal growth in development and in nerve regeneration following nerve injury. This study examines the impact of E-FABP expression in normal neurite extension in differentiating pheochromocytoma cell (PC12) cultures supplemented with selected long chain free fatty acids (LCFFA). We found that E-FABP binds to a broad range of saturated and unsaturated LCFFAs, including those with potential interest for neuronal differentiation and axonal growth such as C22:6n-3 docosahexaenoic acid (DHA), C20:5n-3 eicosapentaenoic acid (EPA), and C20:4n-6 arachidonic acid (ARA). PC12 cells exposed to nerve growth factor (NGFDPC12) exhibit high E-FABP expression that is blocked by mitogen-activated protein kinase kinase (MEK) inhibitor U0126. Nerve growth factor-differentiated pheochromocytoma cells (NGFDPC12) antisense clones (NGFDPC12-AS) which exhibit low E-FABP expression have fewer/shorter neurites than cells transfected with vector only or NGFDPC12 sense cells (NGFDPC12-S). Replenishing NGFDPC12-AS cells with biotinylated recombinant E-FABP (biotin-E-FABP) protein restores normal neurite outgrowth. Cellular localization of biotin-E-FABP in NGFDPC12 was detected mostly in the cytoplasm and in the nuclear region. Treatment of NGFDPC12 with DHA, EPA, or ARA further enhances neurite length but it does not trigger further induction of TrkA or MEK phosphorylation or E-FABP mRNA observed in differentiating PC12 cells without LCFFA supplementation. Significantly, DHA and EPA neurite stimulating effects are higher in NGFDPC12-S than in NGFDPC12-AS cells. These findings are consistent with the scenario that neurite extension of differentiating PC12 cells, including further stimulation by DHA and EPA, requires sufficient cellular levels of E-FABP.     

Localization of epidermal-type fatty acid binding protein in the thymic epithelial cells of mice

The immunoreactivity for epidermal-type fatty acid binding protein of epidermis type (E-FABP) was selectively localized in the epithelial cells of both cortex and medulla of mouse thymus. The cortical epithelial cytoreticulum was clearly visible with the intense immunoreactivity and the immunoreactive cytoreticulum extended intricately throughout the thymic cortex to enclose thymocytes. In the thymic medulla, the immunoreactivity was variable in intensity among the epithelial cells and there was a tendency that epithelial cells containing more numerous tonofilament bundles were less immunoreactive. Considering the possibility that FABPs function as intracellular carriers for unsaturated long chain fatty acids, the present finding suggests that E-FABP in the thymic epithelial cells, especially the cortical ones because of their extensive location, are intimately involved in the metabolic processes of fatty acids including production of bioactive substances, such as prostaglandin and leukotriene, which are known to exert some regulation of thymic immune responses.     

Leukotriene A4, conversion to leukotriene B4 in human T-cell lines

Human T-cell lines (HSB, MOLT-4 and CCRF-CEM) produced leukotriene B4 when incubated with leukotriene A4. The product was characterized by chromatographic properties, UV-spectroscopy and gas chromatography mass spectrometry. About 10 pmol of leukotriene B4 was obtained per 10(6) cells. When incubated with arachidonic acid plus the calcium ionophore A23187 however, no leukotriene B4 was found, indicating that the T-cell lines lack 5-lipoxygenase yet contain LTA4 hydrolase.     

15-Lipoxygenation of leukotriene A(4). Studies Of 12- and 15-lipoxygenase efficiency to catalyze lipoxin formation

The unstable epoxide leukotriene (LT) A(4) is a key intermediate in leukotriene biosynthesis, but may also be transformed to lipoxins via a second lipoxygenation at C-15. The capacity of various 12- and 15-lipoxygenases, including porcine leukocyte 12-lipoxygenase, a human recombinant platelet 12-lipoxygenase preparation, human platelet cytosolic fraction, rabbit reticulocyte 15-lipoxygenase, soybean 15-lipoxygenase and human eosinophil cytosolic fraction, to catalyze conversion of LTA(4) to lipoxins was investigated and standardized against the ability of the enzymes to transform arachidonic acid to 12- or 15-hydroxyeicosatetraenoic acids (HETE), respectively. The highest ratio between the capacity to produce lipoxins and HETE (LX/HETE ratio) was obtained for porcine leukocyte 12-lipoxygenase with an LX/HETE ratio of 0.3. In addition, the human platelet 100000xg supernatant 12-lipoxygenase preparation and the human platelet recombinant 12-lipoxygenase and human eosinophil 100000xg supernatant 15-lipoxygenase preparation possessed considerable capacity to produce lipoxins (ratio 0.07, 0.01 and 0.02 respectively). In contrast, lipoxin formation by the rabbit reticulocyte and soybean 15-lipoxygenases was much less pronounced (LX/HETE ratios <0.002). Kinetic studies of the human lipoxygenases revealed lower apparent K(m) for LTA(4) (9-27 microM), as compared to the other lipoxygenases tested (58-83 microM). The recombinant human 12-lipoxygenase demonstrated the lowest K(m) value for LTA(4) (9 microM) whereas the porcine leukocyte 12-lipoxygenase had the highest V(max). The profile of products was identical, irrespective of the lipoxygenase used. Thus, LXA(4) and 6S-LXA(4) together with the all-trans LXA(4) and LXB(4) isomers were isolated. Production of LXB(4) was not observed with any of the lipoxygenases. The lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate was considerably more efficient to inhibit conversion of LTA(4) to lipoxins, as compared to the inhibitory effect on 12-HETE formation from arachidonic acid (IC(50) 1 and 50 microM, respectively) in the human platelet cytosolic fraction.     

Leukotriene A5 is a substrate and an inhibitor of rat and human neutrophil LTA4 hydrolase

The epoxide 5(S) trans-5,6 oxido, 7,9 trans-11,14,17 cis eicosatetraenoic acid (leukotriene A5) was chemically synthesized and demonstrated to be both a substrate and an inhibitor of partially purified rat and human LTA4 hydrolase. Both rat and human LTA4 hydrolase utilized leukotriene A5 less effectively as a substrate than leukotriene A4. Incubation of leukotriene A5 (10 microM) or leukotriene A4 (10 microM) with rat neutrophils demonstrated formation of 123 pmol LTB5/min/10(7) cells and 408 pmol LTB4/min/10(7) cells respectively. Purified rat neutrophil LTA4 hydrolase incubated with 100 microM leukotriene A5 produced 22 nmol LTB5/min/mg protein and when incubated with 100 microM leukotriene A4 produced 50 nmol LTB4/min/mg protein. Human neutrophil LTA4 hydrolase incubated with 100 microM leukotriene A5 produced 24 nmol LTB5/min/mg protein and when incubated with 100 microM leukotriene A4 produced 52 nmol LTB4/min/mg protein. Leukotriene A5 was an inhibitor of the formation of leukotriene B4 from leukotriene A4 by both the rat and human neutrophil LTA4 hydrolase. Excess leukotriene A5 prevented covalent coupling of [3H] leukotriene A4 to LTA4 hydrolase suggesting inhibition may involve covalent coupling of leukotriene A5 to the LTA4 hydrolase.     

Leukotriene A4 hydrolase is a zinc-containing aminopeptidase

A comparison of amino acid sequences revealed that leukotriene A4 (LTA4) hydrolase is homologous to various types of aminopeptidases. Consistently with the finding, the purified LTA4 hydrolases from both human and guinea pig sources contained equimolar zinc ion, as determined by atomic absorption spectrometry. The enzyme had a significant amount of aminopeptidase activity toward synthetic peptide substrates. Both LTA4 hydrolase and aminopeptidase activities were inhibited by o-phenanthroline, p-chloromercuribenzoic acid, and Leu-thiol with similar IC50 values. Co-purification as well as co-immunoprecipitation of both enzyme activities with an affinity-purified antibody against LTA4 hydrolase strongly suggest that the two enzyme activities reside in a single protein.     

The two calcium-binding proteins, S100A8 and S100A9, are involved in the metabolism of arachidonic acid in human neutrophils.

Recently, we identified the two myeloid related protein-8 (MRP8) (S100A8) and MRP14 (S100A9) as fatty acid-binding proteins (Klempt, M., Melkonyan, H., Nacken, W., Wiesmann, D., Holtkemper, U., and Sorg, C. (1997) FEBS Lett. 408, 81-84). Here we present data that the S100A8/A9 protein complex represents the exclusive arachidonic acid-binding proteins in human neutrophils. Binding and competition studies revealed evidence that (i) fatty acid binding was dependent on the calcium concentration; (ii) fatty acid binding was specific for the protein complex formed by S100A8 and S100A9, whereas the individual components were unable to bind fatty acids; (iii) exclusively polyunsaturated fatty acids were bound by S100A8/A9, whereas saturated (palmitic acid, stearic acid) and monounsaturated fatty acids (oleic acid) as well as arachidonic acid-derived eicosanoids (15-hydroxyeicosatetraenoic acid, prostaglandin E(2), thromboxane B(2), leukotriene B(4)) were poor competitors. Stimulation of neutrophil-like HL-60 cells with phorbol 12-myristate 13-acetate led to the secretion of S100A8/A9 protein complex, which carried the released arachidonic acid. When elevation of intracellular calcium level was induced by A23187, release of arachidonic acid occurred without secretion of S100A8/A9. In view of the unusual abundance in neutrophilic cytosol (approximately 40% of cytosolic protein) our findings assign an important role for S100A8/A9 as mediator between calcium signaling and arachidonic acid effects. Further investigations have to explore the exact function of the S100A8/A9-arachidonic acid complex both inside and outside of neutrophils.     

Effects of a Synthetic bFGF Antagonist Peptide on the Proteome of 3T3 Cells Stimulated with bFGF

It has been known that basic fibroblast growth factor (bFGF) plays an important role in tumor progression mainly due to its strong mitogenic activity. Antagonists targeting bFGF have been considered a potential strategy for cancer therapy via inhibiting cell proliferation induced by bFGF. We have previously obtained a high-affinity bFGF-binding peptide (named as P7) with strong inhibitory activity against bFGF-induced cell proliferation from the phage display random heptapeptide library. The aim of the present investigation was to synthesize the peptide P7 by solid phase method and explore the mechanisms of the inhibitory effect of P7-targeting bFGF stimulation on Balb/c 3T3 cells via proteomic analysis. Seven differentially expressed protein were identified, among which four were decreased by bFGF stimulation alone and increased by addition of P7, the other three were up-regulated by bFGF treatment alone and down-regulated by addition of P7. Among the identified proteins, epidermal fatty acid-binding protein (E-FABP) and low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) take part in the regulation of cell proliferation. The results suggested P7 inhibited the bioactivities of bFGF possibly via influencing the expression of cellular proteins related to cell proliferation.     

Lipid metabolism and adipokine levels in fatty acid-binding protein null and transgenic mice

Fatty acid-binding proteins (FABPs) facilitate the diffusion of fatty acids within cellular cytoplasm. Compared with C57Bl/6J mice maintained on a high-fat diet, adipose-FABP (A-FABP) null mice exhibit increased fat mass, decreased lipolysis, increased muscle glucose oxidation, and attenuated insulin resistance, whereas overexpression of epithelial-FABP (E-FABP) in adipose tissue results in decreased fat mass, increased lipolysis, and potentiated insulin resistance. To identify the mechanisms that underlie these processes, real-time PCR analyses indicate that the expression of hormone-sensitive lipase is reduced, while perilipin A is increased in A-FABP/aP2 null mice relative to E-FABP overexpressing mice. In contrast, de novo lipogenesis and expression of genes encoding lipoprotein lipase, CD36, long-chain acyl-CoA synthetase 5, and diacylglycerol acyltransferase are increased in A-FABP/aP2 null mice relative to E-FABP transgenic animals. Consistent with an increase in de novo lipogenesis, there was an increase in adipose C16:0 and C16:1 acyl-CoA pools. There were no changes in serum free fatty acids between genotypes. Serum levels of resistin were decreased in the E-FABP transgenic mice, whereas serum and tissue adiponectin were increased in A-FABP/aP2 null mice and decreased in E-FABP transgenic animals; leptin expression was unaffected. These results suggest that the balance between lipolysis and lipogenesis in adipocytes is remodeled in the FABP null and transgenic mice and is accompanied by the reprogramming of adipokine expression in fat cells and overall changes in plasma adipokines.     

Phospholipid bilayers enhance the stability of leukotriene A4 and epoxytetraenes: stabilization of eicosanoids by liposomes

The hydrolysis of leukotriene A4 and two epoxytetraenes was examined in the presence and absence of liposomes. When added to liposomes in suspension, the stability of LTA4 was increased in a time- and dose-dependent fashion. At 10 min, the half-life of LTA4 was increased 67.1 +/- 6.8% in the presence of liposomes which was comparable to that observed with albumin (10 mg/ml): 68.3 +/- 6.9%. Phosphatidylcholine, in a non-bilayer configuration, was also effective in enhancing the half-life of LTA4, albeit to a lesser extent than liposomes. At equal molar concentrations, the enhanced stability of eicosanoid epoxides with liposomes gave a rank order with leukotriene A4 greater than 5(6)epoxytetraene greater than 14(15)epoxytetraene. Results indicate that phospholipid bilayers can protect leukotriene A4 and 5(6)epoxytetraene from non-enzymatic hydrolysis. Moreover, they suggest that the biological half-life of intermediates involved in the formation of both leukotrienes and lipoxins can be increased by their association with membranes.     

Tetraene and pentaene leukotrienes: selective production from murine mastocytoma cells after dietary manipulation

A neoplastic mast cell tumor was grown in mice which had been raised since birth on a diet enriched with eicosapentaenoic acid. Intact harvested mastocytoma cells were stimulated with calcium ionophore A23187 to produce lipoxygenase products from the polyunsaturated fatty acids liberated from the cellular membranes. Leukotriene B4, B5, C4, and C5 were isolated and characterized by HPLC retention time, ultraviolet absorption spectrometry and mass spectrometry. The arachidonic acid content of the mast cell tumor lipids was altered from 9.2 to 3.9 mole % while eicosapentaenoic acid increased from 0.5 to 4.5 mole % in response to the fish oil-supplemented diet. The relative amounts of arachidonic and eicosapentaenoic acids (3.9 and 4.5 mole % respectively) were associated with similar amounts of LTB4 and LTB5 synthesized by the cells. These results suggest that the epoxide leukotriene (LIA) derivative can be made efficiently from either arachidonic or eicosapentaenoic acids when both are present in cellular lipids. In contrast, the ratio of LTC4 to LTC5 (10 to 1) indicates that the reaction of LTA with glutathione may be critically dependent upon the structure of the unsaturated fatty acid with the ratio of LTC4/LTB4 (2.0) more than 10 times greater than that (0.16) for LTC5/LTB5.     

Formation of murine macrophage-derived 5-oxo-7-glutathionyl-8,11,14-eicosatrienoic acid (FOG7) is catalyzed by leukotriene C4 synthase.

5-Oxo-7-glutathionyl-8,11,14-eicosatrienoic acid (FOG(7)), a biologically active glutathione (GSH) adduct of the eicosanoid 5-oxo-eicosatrienoic acid (5-oxoETE), is the major metabolite formed within the murine peritoneal macrophage. The conjugation of GSH to electrophilic 5-oxoETE in vitro was found to be catalyzed by both soluble glutathione S-transferase and membrane-bound leukotriene C(4) (LTC(4)) synthase. The cytosolic glutathione S-transferase-catalyzed products were not biologically active; however, the adduct formed from recombinant LTC(4) synthase had identical mass spectrometric properties and biological activity to the macrophage-derived FOG(7). The biosynthesis of FOG(7) in the macrophage was inhibited by MK-886, a known inhibitor of LTC(4) synthase, suggesting that this nuclear membrane-bound enzyme might be responsible for GSH conjugation to 5-oxoETE in the intact cell. Subcellular fractionation revealed that the microsomal fraction from the murine macrophage contained the enzyme responsible for FOG(7) biosynthesis. Western blot analysis confirmed the presence of LTC(4) synthase in the microsomal fraction that did not catalyze conjugation of GSH to 1-chloro-2,4-dinitrobenzene, indicating an absence of microsomal glutathione S- transferase activity. These results suggest that LTC(4) synthase, thought to be specific for the conjugation of GSH to LTA(4), can also recognize 5-oxoETE as an electrophilic substrate.     

Mass spectrometric quantitation of deoxyguanosine and leukotriene A4-deoxyguanosine adducts of DNA

An assay was developed using electrospray ionization negative ion tandem mass spectrometry (MS) to identify and quantitate the major product in the reaction of leukotriene A(4) (LTA(4)) with deoxyguanosine (dGuo). A second quantitative assay was established using the same separation and detection techniques to determine the amount of dGuo isolated from enzymatically processed DNA. The amount of LTA(4)-dGuo adduct could then be analytically determined in DNA samples and normalized to the amount of dGuo that had been simultaneously derived from the DNA sample. Stable isotope-labeled internal standards used for these quantitative assays were readily synthesized from isotopically labeled [(15)N(5)(13)C(10)]deoxyguanosine triphosphate and analyzed for isotopic purity using MS. A comparison of fragment ions formed from stable isotope analogs of dGuo revealed the loss of deoxyribose and secondarily the loss of a series of stable neutral small molecules in a fashion similar to patterns described previously for the collisional fragmentation of protonated guanine determined by positive ion fast atom bombardment/MS/MS. The combined quantitative assays were used for the determination of the amount of endogenously formed LTA(4)-dGuo adducts observed in DNA when isolated human neutrophils that had been incubated with arachidonic acid were stimulated with calcium ionophore to initiate leukotriene biosynthesis.     

Inhibition by adenosine 3':5'-monophosphate of eicosanoid and platelet-activating factor biosynthesis in the mouse PT-18 mast cell.

A mouse spleen-derived mast cell line (PT-18) was employed to examine the mechanisms of adenosine 3':5'-monophosphate (cAMP)-mediated inhibition of antigen-induced lipid mediator biosynthesis. Specifically, we tested the hypothesis that increasing cAMP in mast cells inhibits lipid mediator biosynthesis by a mechanism independent of effects on histamine release (degranulation) or changes in cytosolic calcium concentration. Forskolin inhibited antigen-induced prostaglandin D2 (PGD2), leukotriene C4 (LTC4), and leukotriene B4 (LTB4) production by 30-50%. In contrast, forskolin had no inhibitory effect on antigen-induced increases in cytosolic calcium concentration, as monitored by the calcium indicator fura-2, or histamine release from the cells. The combination of the phosphodiesterase inhibitor isobutylmethylxanthine with forskolin inhibited the antigen-induced production of PGD2 and LTC4 by 90-100% and histamine release by about 60%. These responses were accompanied by a virtual abolition of the antigen-induced increase in cytosolic calcium. To test further the hypothesis that increasing cAMP can lead to inhibition of lipid mediator biosynthesis in the absence of effects on cytosolic calcium, we employed the calcium ionophores A23187 and ionomycin. Forskolin alone or in combination with isobutylmethylxanthine had no effect on ionophore-induced increases in cytosolic calcium but effectively inhibited leukotriene biosynthesis. In addition, increasing cyclic AMP led to an inhibition of ionophore-induced production of platelet-activating factor and liberation of arachidonic acid. These data suggest that a relatively modest increase in cAMP-dependent protein kinase activity in mast cells leads to inhibition of the lipase-catalyzed cleavage of arachidonic acid from membrane phospholipids in the absence of measurable effects on either histamine release or changes in cytosolic calcium concentration. This effect results in a selective inhibition of the biosynthesis of lipid mediators including LTC4, LTB4, PGD2, and platelet-activating factor.     

Cardiac fatty acid-binding proteins. Isolation and characterization of the mitochondrial fatty acid-binding protein and its structural relationship with the cytosolic isoforms

In the course of our studies on the structural diversity of the isoforms of cardiac fatty acid-binding proteins (cFABPs), a cardiac-type FABP from the matrix of bovine heart mitochondria was purified to homogeneity and obtained as a single 15-kDa protein with an isoelectric point of 4.9. The primary structures of this protein and of the two isoforms isolated from the cytosol (pI4.9-cFABP and pI 5.1-cFABP) were investigated by means of plasma desorption mass spectrometry and sequencing of peptides. All three proteins are amino-terminally blocked with an acetyl group and shown to be colinear with the sequence deduced from a cDNA clone for bovine heart fatty acid-binding protein (Billich, S., Wissel, T., Kratzin, H., Hahn, U., Hagenhoff, B., Lezius, A. G., and Spener, F. (1988) Eur. J. Biochem. 175, 549-556) except for the residue at position 98. This residue is demonstrated to be the molecular origin of bovine cFABP isoforms since pI 5.1-cFABP contains Asn98 in accordance with the sequence derived from the cDNA, whereas in pI 4.9-cFABP, this position is occupied by Asp98. Moreover, mitochondrial FABP is identical to pI 4.9-cFABP. Molecular masses of pI 4.9-cFABP (14,679 +/- 10 Da) and pI 5.1-cFABP (14,678 +/- 20 Da) determined by plasma desorption mass spectrometry coincide with that calculated from the cDNA (14,673 Da). Hence, residues linked to these proteins by posttranslational modification are not present, and the Asn-Asp exchange is the sole origin of heterogeneity of mitochondrial and cytosolic fatty acid-binding proteins from bovine heart.