Inhibition of the formation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid from arachidonic acid in polymorphonuclear leukocytes by various coumarins

The effects of various coumarins (i.e. esculetin, daphnetin and fraxetin) on the formation of the 5-lipoxygenase product, 5-HETE, and the cyclooxygenase product, HHT, were studied. Esculetin (6,7-dihydroxycoumarin) was found to inhibit the formation of 5-HETE more strongly than HHT; its concentrations for 50% inhibition (IC50) were 1.46 +/- 1.02 microM for the formation 5-HETE and 57.3 +/- 17.3 microM for the formation of HHT. Daphnetin (7,8-dihydroxycoumarin) and fraxetin (6-methoxy-7,8-dihydroxycoumarin) also inhibited the formation of the 5-lipoxygenase product, 5-HETE, and the cyclooxygenase product, HHT; their IC50 values were, respectively, 6.90 +/- 2.07 microM and 2.57 +/- 0.088 microM for the formation of 5-HETE and 139.0 +/- 30.0 microM and 532.5 +/- 33.0 microM for the formation of HHT. The monohydroxy coumarin derivatives umbelliferone (7-hydroxycoumarin) and scopoletin (6-methoxy-7-hydroxycoumarin) and the coumarin glucosides fraxin (6-methoxy-7,8-dihydroxycoumarin 8-O-D-glucoside) and esculin (6,7-dihydroxycoumarin 6-O-D-glucoside) also inhibited the formation of 5-HETE, though less strongly. 4-Hydroxycoumarin and coumarin had no effect on either 5-lipoxygenase or cyclooxygenase at concentrations of up to 1 mM. Esculetin inhibited the formation of 5-HETE noncompetitively. In contrast, the dimethoxycoumarin fraxidin (6,8-dimethoxy-7-hydroxycoumarin) inhibited the formation of HHT more strongly than the formation of 5-HETE at a concentration of 1 mM.     

Biosynthesis of 5-oxo-6,8,11,14-eicosatetraenoic acid from 5-hydroperoxyeicosatetraenoic acid in the murine macrophage

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a metabolite of arachidonic acid shown to possess important biological activities within different cell types. In the neutrophil, a specific NADP(+)-dependent dehydrogenase utilizes 5-lipoxygenase-derived 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5(S)-HETE) as the required substrate. In the present study, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HpETE), rather than 5-HETE, was found to be the biosynthetic precursor of 5-oxo-ETE in the murine macrophage. The macrophage was not able to convert 5-HETE into 5-oxo-ETE even when preincubated with phorbol ester or with other lipid hydroperoxides. The factor responsible for the conversion of 5-HpETE into 5-oxo-ETE was found predominantly in the cytosolic fraction of the macrophage, with an approximate molecular weight of 50,000-60,000, as assessed by size exclusion chromatography. Formation of 5-oxo-ETE was rapid and the catalytic protein was found to have an apparent K(m) of 5.3 microM for the eicosanoid. Furthermore, the protein could efficiently utilize 5(R,S)-HpETE as substrate and was heat and protease labile. This novel pathway of 5-oxo-ETE biosynthesis in the murine macrophage was consistent with reduction of a 5-hydroperoxy group to an intermediate alkoxy radical that could be subsequently oxidized to the 5-oxo product. Such a mechanism would enable racemic 5-HpETE, derived from free radical oxidation of arachidonic acid, to be efficiently converted into this potent chemotactic eicosanoid.     

Metabolism of 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid and other 5(S)-hydroxyeicosanoids by a specific dehydrogenase in human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNL) convert 6-trans isomers of leukotriene B4 (LTB4) to dihydro metabolites (Powell, W.S., and Gravelle, F. (1988) J. Biol. Chem. 263, 2170-2177). In the present study we investigated the mechanism for the initial step in the formation of these products. We found that the 1,500 x g supernatant fraction from human PMNL converts 12-epi-6-trans-LTB4 to its 5-oxo metabolite which was identified by mass spectrometry and UV spectrophotometry. The latter compound was subsequently converted to the corresponding dihydro-oxo product, which was further metabolized to 6,11-dihydro-12-epi-6-trans-LTB4, which was the major product after longer incubation times. The 5-hydroxyeicosanoid dehydrogenase activity is localized in the microsomal fraction and requires NADP+ as a cofactor. These experiments therefore suggest that the initial step in the formation of dihydro metabolites of 6-trans isomers of LTB4 is oxidation of the 5-hydroxyl group by a microsomal dehydrogenase. Studies with a variety of substrates revealed that the microsomal dehydrogenase in human PMNL oxidizes the hydroxyl groups of a number of other eicosanoids which contain a 5(S)-hydroxyl group followed by a 6-trans double bond. There is little or no oxidation of hydroxyl groups in the 8-, 9-, 11-, 12-, or 15-positions of eicosanoids, or of the 5-hydroxyl group of LTB4, which has a 6-cis rather than a 6-trans double bond. The preferred substrate for this enzyme is 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5(S)-HETE) (Km, 0.2 microM), which is converted to 5-oxo-6,8,11,14-eicosatetraenoic acid. Unlike 5(S)-HETE, 5(R)-HETE is a poor substrate for the 5(S)-hydroxyeicosanoid dehydrogenase, indicating that in addition to exhibiting a high degree of positional specificity, this enzyme is also highly stereospecific. In addition to 5(S)-HETE and 6-trans isomers of LTB4, 5,15-diHETE is also a good substrate for this enzyme, being converted to 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid (5-oxo-15-hydroxy-ETE). The oxidation of 5(S)-HETE to 5-oxo-ETE is reversible since human PMNL microsomes stereospecifically reduce 5-oxo-ETE to the 5(S)-hydroxy compound in the presence of NADPH. 5-Oxo-ETE is formed rapidly from 5(S)-HETE by intact human PMNL, but because of the reversibility of the reaction, its concentration only reaches about 25% that of 5(S)-HETE.     

Oxidative stress stimulates the synthesis of the eosinophil chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid by inflammatory cells

5-Oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid) is a highly potent granulocyte chemoattractant that acts through a selective G-protein coupled receptor. It is formed by oxidation of the 5-lipoxygenase product 5-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) by 5-hydroxyeicosanoid dehydrogenase (5-HEDH). Although leukocytes and platelets display high microsomal 5-HEDH activity, unstimulated intact cells do not convert 5-HETE to appreciable amounts of 5-oxo-ETE. To attempt to resolve this dilemma we explored the possibility that 5-oxo-ETE synthesis could be enhanced by oxidative stress. We found that hydrogen peroxide and t-butyl hydroperoxide strongly stimulate 5-oxo-ETE formation by U937 monocytic cells. This was dependent on the GSH redox cycle, as it was blocked by depletion of GSH or inhibition of glutathione reductase and mimicked by oxidation of GSH to GSSG by diamide. Glucose inhibited the response to H2O2 through its metabolism by the pentose phosphate pathway, as its effect was reversed by the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. 5-Oxo-ETE synthesis was also strongly stimulated by hydroperoxides in blood monocytes, lymphocytes, and platelets, but not neutrophils. Unlike monocytic cells, lymphocytes and platelets were resistant to the inhibitory effects of glucose. 5-Oxo-ETE synthesis following incubation of peripheral blood mononuclear cells with arachidonic acid and calcium ionophore was also strongly enhanced by t-butyl hydroperoxide. Oxidative stress could act by depleting NADPH, resulting in the formation NADP+, the cofactor for 5-HEDH. This is opposed by the pentose phosphate pathway, which converts NADP+ back to NADPH. Oxidative stress could be an important mechanism for stimulating 5-oxo-ETE production in inflammation, promoting further infiltration of granulocytes into inflammatory sites.     

Quantitative analysis of 5-oxo-6,8,11,14-eicosatetraenoic acid by electrospray mass spectrometry using a deuterium-labeled internal standard

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a metabolite of arachidonic acid formed by the 5-lipoxygenase pathway, is a potent eosinophil chemoattractant that may be an important mediator in asthma. To further investigate the physiological and pathological roles of 5-oxo-ETE we have developed a mass spectrometric assay employing a tetradeuterated analog (5-oxo-[11,12,14,15-(2)H]ETE) as an internal standard. Collision-induced dissociation of the quasimolecular anion of 5-oxo-[11,12,14,15-(2)H]ETE (m/z 321) resulted in the formation of a major ion at m/z 207 that retained all four deuterium atoms. Measurement of the ratio of ions at m/z 203 (endogenous 5-oxo-ETE) and m/z 207 permitted quantitation of this compound by liquid chromatography-mass spectrometry-mass spectrometry using multiple reaction monitoring. The resulting assay was highly sensitive (< or =20 pg/sample) and selective, enabling detection of the amount of 5-oxo-ETE produced by as few as 10,000 neutrophils. This assay should permit measurement of 5-oxo-ETE in biological fluids, enabling evaluation of its role in asthma and other inflammatory diseases.     

Transfomration of arachidonic acid into 12-hydroxy-5,8,10,14-eicosatetraenoic acid by mouse peritoneal macrophages.

Mouse peritoneal macrophages were incubated at 37 degrees C for 30 min with arachidonic acid (all-cis-5,8,11,14-eicosatetraenoic acid). Oxygenation of arachidonic acid in mouse peritoneal macrophages occurs by two major pathways: fatty acid cyclooxygenase and lipoxygenase. The major metabolite of the latter is 12-hydroxy-5,8,10,14-eicosatetraenoic acid which was identified by gas liquid chromatography on high resolution glass capillary column and mass spectrometry.     

Biosynthesis and metabolism of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid by human umbilical vein endothelial cells

Incubation of cultured human umbilical vein endothelial cells with [1-14C]arachidonic acid, followed by reverse-phase high-pressure liquid chromatography analysis, results in the appearance of two principal radioactive products besides 6-keto-prostaglandin F1 alpha. The first peak is 12-L-hydroxy-5,8,10-heptadecatrienoic acid, a hydrolysis product of the prostaglandin endoperoxide. The second peak was esterified, converted to the trimethylsilyl ether derivative, and analyzed by gas chromatography-mass spectrometry and shown to be the lipoxygenase product 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). Incubation of the 15-HETE precursor 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) with endothelial cells results in the formation of four distinct UV absorbing peaks. UV and gas chromatography-mass spectrometry analysis showed these peaks to be 8,15(S)-dihydroxy-5,8,11,13-eicosatetraenoic acids (8,15-diHETE) differing only in their hydroxyl configuration and cis trans double-bond geometry. Formation of 8,15-diHETE molecules suggests the prior formation of the unstable epoxide molecule 14(S),15(S)-trans-oxido-5,8-Z-14,15-leukotriene A4 or an attack at C-10 of 15-HPETE by an enzyme with mechanistic features in common with a 12-lipoxygenase. The observation that endothelial cells can synthesize both 15-HETE and 8,15-diHETE molecules suggests that this cell type contains both a 15-lipoxygenase and a system that can synthesize 14,15-leukotriene A4.     

5-oxo-6,8,11,14-eicosatetraenoic acid stimulates the release of the eosinophil survival factor granulocyte/macrophage colony-stimulating factor from monocytes

Allergic diseases such as asthma are characterized by tissue eosinophilia induced by the combined effects of chemoattractants and cytokines. Lipid mediators are a major class of endogenous chemoattractants, among which 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is the most potent for human eosinophils. In this study, we investigated the effects of 5-oxo-ETE on eosinophil survival by flow cytometry. We found that this compound could promote eosinophil survival in the presence of small numbers of contaminating monocytes, but not in their absence. The conditioned medium from monocytes treated for 24 h with 5-oxo-ETE also strongly promoted eosinophil survival, whereas the medium from vehicle-treated monocytes had no effect. An antibody against the granulocyte/macrophage colony-stimulating factor (GM-CSF) completely blocked the response of eosinophils to the conditioned medium from 5-oxo-ETE-treated monocytes, whereas an antibody against interleukin-5 had no effect. Furthermore, 5-oxo-ETE stimulated the release of GM-CSF from cultured monocytes in amounts compatible with eosinophil survival activity, with a maximal effect being observed after 24 h. This effect was concentration-dependent and could be observed at concentrations in the picomolar range. 5-Oxo-ETE and leukotriene B(4) had similar effects on GM-CSF release at low concentrations, but 5-oxo-ETE induced a much stronger response at concentrations of 10 nm or higher. This is the first report that 5-oxo-ETE can induce the release of any cytokine, suggesting that it could be an important mediator in allergic and other inflammatory diseases due both to its chemoattractant properties and to its potent effects on the synthesis of the survival factor GM-CSF.     

Identification of 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid as a novel and potent human eosinophil chemotactic eicosanoid.

Incubation of human eosinophils with arachidonic acid led to the formation of a novel and potent eosinophil chemotactic lipid (ECL) (Morita, E., Schr?der, J.-M., and Christophers, E. (1990) J. Immunol. 144, 1893-1900). To test the working hypothesis of whether ECL could have been formed via eosinophil-arachidonic acid 15-lipoxygenase we investigated whether other arachidonic acid 15-lipoxygenases such as soybean lipoxygenase I catalyze formation of a similar ECL. In the presence of hemoproteins and soybean lipoxygenase I arachidonic acid is converted to an ECL, which has physicochemical properties similar to those found for the eosinophil-derived ECL. Purification of this ECL by high performance liquid chromatography revealed that ECL is structurally different from well known eosinophil chemotactic eicosanoids such as leukotriene B4, 5,15-(6E,8Z,11Z,13E)-dihydroxyeicosatetraenoic acid (5,15-diHETE), and (8S,15S)-(5Z,9E,11Z,13E)-dihydroxyeicosatetra eno ic acid ((8S,15S)-diHETE). UV spectra of this ECL with absorbance maxima at 230 and 278 nm revealed the presence of two independent chromophores such as a conjugated oxodiene and a conjugated diene. Catalytic hydrogenation of ECL methyl ester led to the formation of 5,15-dihydroxyarachidic acid methyl ester. Reduction of ECL with sodium borohydride produced a product which is identical with authentic (5S,15S)-(6E,8Z,11Z,13E)-diHETE. Formation of an ECL monomethoxime derivative supports the conclusion that this highly potent eosinophil chemotactic eicosanoid is structurally identical with 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid.     

Effect of ethanolamine on choline uptake and incorporation into phosphatidylcholine in human Y79 retinoblastoma cells

The effect of physiological concentrations of ethanolamine on choline uptake and incorporation into phosphatidylcholine was investigated in human Y79 retinoblastoma cells, a multipotential, undifferentiated retinal cell line that has retained many neural characteristics. These cells have a high-affinity uptake system for choline, and the majority of the choline taken up was incorporated into phosphatidylcholine via the CDP-choline pathway. The presence of extracellular ethanolamine significantly decreased high-affinity choline uptake and, subsequently, the amount of choline incorporated into phosphatidylcholine. When 100 mumol/L ethanolamine was added, there was a decrease of about 8% in the phosphatidylcholine content. Ethanolamine had no effect on choline incorporation into phosphatidylcholine, however, once choline was taken up by the cell. The K'M and V'max for high-affinity choline uptake was increased from 0.93 to 9.74 microM and 19.60 to 79.25 pmol/min per mg protein, respectively, by the presence of 25 mumol/L ethanolamine. In contrast, 25 mumol/L choline had no effect on the kinetic parameters of high-affinity ethanolamine uptake. Therefore, the reduction in high-affinity choline transport by ethanolamine apparently is not simply due to competitive inhibition. 2,2-Dimethylethanolamine and 2-methylethanolamine both reduced choline uptake to a greater extent than ethanolamine. However, because these compounds exist at much lower concentrations than ethanolamine, they probably have little physiological influence. These results suggest that changes in ethanolamine concentration within the physiologic range can regulate the synthesis and content of phosphatidylcholine in a neural cell by influencing the uptake of choline.     

Specific incorporation of 5-fluorocytidine into Escherichia coli RNA

RNAs isolated from Escherichia coli B grown in the presence of 5-fluorouracil have high levels of the analog replacing uridine and uridine-derived modified nucleosides. Cytidine has also been shown to be replaced in these RNAs by 5-fluorocytidine, a metabolic product of 5-fluorouracil, but to a considerably lesser extent. When 5-fluorocytidine is added to cultured of E. coli B little 5-fluorocytidine (0.20 mol%) is incorporated into cellular RNAs because of the active cytosine/cytidine deaminase activities. Addition of the cytidine deaminase inhibitor tetrahydrouridine (70 micrograms/ml) increases 5-fluorocytidine incorporation to about 3 mol% in tRNAs, but does not eliminate 5-fluorouridine incorporation. E. coli mutants lacking cytosine/cytidine deaminase activities are able to more than double the extent of 5-fluorocytidine incorporation into their transfer and ribosomal RNAs, replacing cytidine with no detectable 5-fluorouridine incorporation. Levels of 5-methyluridine, pseudouridine and dihydrouridine in tRNAs are not affected. These fluorocytidine-containing tRNAs show amino acid-accepting activities similar to control tRNAs. Fluorocytidine was found to be quite susceptible to deamination under alkaline conditions. Its conversion to primarily 5-fluorouridine follows pseudo-first-order reaction kinetics with a half-life of 10 h in 0.3 M KOH at 37 degrees C. This instability in alkali probably explains why 5-fluorocytidine was not found earlier in RNAs isolated from cells treated with 5-fluorouridine, since most early RNA hydrolyses were carried out in alkali. It may also explain the mild mutagenic properties observed in some systems following 5-fluorouridine treatment. Initial 19F-NMR measurements in fluorocytidine-containing tRNAs indicate that this modified tRNA may be useful in future structural studies of tRNAs and in probing tRNA-protein complexes.     

Metabolism of 15-hydroxy-5,8,11,13-eicosatetraenoic acid by MOLT-4 cells and blood T-lymphocytes

MOLT-4 lymphocytes metabolize 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) via beta-oxidation with retention of the hydroxyl group at the omega 6-carbon atom. 15-HETE oxidation is accompanied by the time-dependent accumulation of both beta-hydroxy acids and metabolites produced by repetitive cycles of the beta-oxidation spiral. Detection of 7-hydroxy-5-dodecenoic acid shows that these cells continue to beta-oxidize the substrate when the conjugated diene is allylic to a hydroxyl group. When 15-HETE was the substrate, it was also possible to detect 12-hydroxy-5,8,10-heptadecatrien-1-al and 3,15-dihydroxy-8,11,13-eicosatrienoic acid. The former product may be produced by alpha-oxidation of 13-hydroxy-6,9,11-octadecatrienoic acid followed by its decarboxylation. Detection of a 20-carbon metabolite, lacking a double bond at position 5, suggests that an intermediate of beta-oxidation was used as a substrate for chain elongation. When 13-hydroxy-6,9,11-octadecatrienoic acid was used as a substrate, it was indeed possible to detect 3,15-dihydroxy-8,11,13-eicosatrienoic acid as well as 15-hydroxy-8,11,13-eicosatrienoic acid. In addition, 13-hydroxy-6,9,11-octadecatrienoic acid was a precursor for the biosynthesis of both 14-hydroxy-7,10,12-nonadecatrien-1-al and 1,14-dihydroxy-7,10,12-nonadecatriene. These studies with MOLT-4 cells as well as with T-lymphocytes isolated from blood show that products of the 15-lipoxygenase pathway are metabolized with the accumulation of a variety of compounds. Since 15-HETE has been implicated as a modulator of T-cell function, these findings raise the possibility that the newly described metabolites may be involved in regulating lymphocyte function.     

Specific herpes simplex virus-induced incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine into deoxyribonucleic acid.

5-Iodo-5'-amino-2',5'-dideoxyuridine (AIdUrd) is a novel thymidine analog which inhibits herpes simplex virus, type 1 (HS-1 virus) replication in the absence of detectable host toxicity. When murine, simian, or human cells in culture are treated with [125I]AIdUrd for up to 24 hours essentially none of the nucleoside becomes cell-associated. In contrast, upon HS-1 virus infection significant radiolabel is detected in both nucleotide pools and in DNA. The major acid-soluble metabolite has been shown by enzymic and chromatographic analysis to be the 5'-triphosphate of AIdUrd. DNA from HS-1 virus-infected Vero cells labeled with [14C]thymidine, 5-[125I]iodo-2'-deoxyuridine (IdUrd), or [125I]AIdUrd was isolated by buoyant density centrifugation and subjected to digestion by pancreatic DNase I, spleen DNase II, micrococcal nuclease, spleen, and venom phosphodiesterases. Analysis of the digestion products clearly indicate that AIdUrd is incorporated internally into the DNA structure. DNA containing AIdUrd therefore contains phosphoramidate (P-N) bonds, known to be extremely acid-labile. The selective HS-1 virus-induced phosphorylation of AIdUrd and its subsequent incorporation into DNA may account for the unique biological activity of the AIdUrd nucleoside.     

12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid is a chemoattractant fo human polymorphonuclear leucocytes in vitro

Increased amounts of 12-hydroxy - 5,8,10,14-eicosatetraenoic acid (12-HETE) are found in the lesional skin of patients with the skin disease psoriasis when compared to clinically normal skin. Stereochemical analysis has recently shown that the 12-HETE present in lesional psoriatic scale is the (R), and not the (S) hydroxyl enantiomer, produced by platelets. Since the chemoattractant activity of 12(R)-HETE has not previously been described, the (R) and (S) hydroxyl enantiomers of 12-HETE have now been synthesised and their chemokinetic activity compared in vitro. 12(R)-HETE, was more potent than 12(S)-HETE as a chemokinetic agent for human polymorphonuclear leucocytes but 2000 times less potent than leukotriene B₄. In contrast to results obtained with the 12-HETE enantiomers, the chemoattractant compound 5(S)-HETE was found to be more potent than the 5(R) hydroxyl enantiomer. Thus, the configuration of the hydroxyl group appears to be of importance to the chemokinetic activity of the HETEs, and the increased potency of the 12(R) enantiomer may enhance its significance as a mediator of inflammation in psoriasis.     

Calcium ionophore A 23187 induces arachidonic acid release from phosphatidylcholine in cultured human endothelial cells

Cultured endothelial cells from human umbilical vein were incubated with (³H)arachidonic acid for 24 hours. The label was incorporated into phospholipids (79.3 %), neutral lipids (15.6 %) and non-esterified fatty acids (4.7 %). Upon challenge with the calcium ionophore A 23187, 5.3 % of the total radioactivity were found in supernatant and corresponded to 6-keto-prostaglandin F_1α (1.6 %) and free arachidonic acid (3.7 %). This release was accompanied by a concomitant and selective decrease of phosphatidylcholine. It is concluded that the entry of calcium promoted by A 23187 activates a phospholipase A₂ regulating the availability of arachidonic acid to the prostacyclin synthetase.     

Mechanism of uptake and incorporation of the non-human sialic acid N-glycolylneuraminic acid into human cells

N-Glycolylneuraminic acid (Neu5Gc) is a widely expressed sialic acid in mammalian cells. Although humans are genetically deficient in producing Neu5Gc, small amounts are present in human cells in vivo. A dietary origin was suggested by human volunteer studies and by observing that free Neu5Gc is metabolically incorporated into cultured human carcinoma cells by unknown mechanisms. We now show that free Neu5Gc uptake also occurs in other human and mammalian cells. Inhibitors of certain non-clathrin-mediated endocytic pathways reduce Neu5Gc accumulation. Studies with human mutant cells show that the lysosomal sialic acid transporter is required for metabolic incorporation of free Neu5Gc. Incorporation of glycosidically bound Neu5Gc from exogenous glycoconjugates (relevant to human gut epithelial exposure to dietary Neu5Gc) requires the transporter as well as the lysosomal sialidase, which presumably acts to release free Neu5Gc. Thus, exogenous Neu5Gc reaches lysosomes via pinocytic/endocytic pathways and is exported in free form into the cytosol, becoming available for activation and transfer to glycoconjugates. In contrast, N-glycolylmannosamine (ManNGc) apparently traverses the plasma membrane by passive diffusion and becomes available for conversion to Neu5Gc in the cytosol. This mechanism can also explain the metabolic incorporation of chemically synthesized unnatural sialic acids, as reported by others. Finally, to our knowledge, this is the first example of delivery to the cytosol of an extracellular small molecule that cannot cross the plasma membrane, utilizing fluid pinocytosis and a specific lysosomal transporter. The approach could, thus, potentially be generalized to any small molecule that has a specific lysosomal transporter but not a plasma membrane transporter.     

12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid is a chemoattractant for human polymorphonuclear leucocytes in vitro

Increased amounts of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) are found in the lesional skin of patients with the skin disease psoriasis when compared to clinically normal skin. Stereochemical analysis has recently shown that the 12-HETE present in lesional psoriatic scale is the (R), and not the (S) hydroxyl enantiomer, produced by platelets. Since the chemoattractant activity of 12(R)-HETE has not previously been described, the (R) and (S) hydroxyl enantiomers of 12-HETE have now been synthesised and their chemokinetic activity compared in vitro. 12(R)-HETE, was more potent than 12(S)-HETE as a chemokinetic agent for human polymorphonuclear leucocytes but 2000 times less potent than leukotriene B4. In contrast to results obtained with the 12-HETE enantiomers, the chemoattractant compound 5(S)-HETE was found to be more potent than the 5(R) hydroxyl enantiomer. Thus, the configuration of the hydroxyl group appears to be of importance to the chemokinetic activity of the HETEs, and the increased potency of the 12(R) enantiomer may enhance its significance as a mediator of inflammation in psoriasis.     

In vitro study of docosahexaenoic acid incorporation into phosphatidylcholine by enzymes of rat heart

Several studies have shown that in animals fed fish oils, docosahexaenoic acid (DHA) is incorporated into cardiac phosphatidylcholines (PC) mainly at the expense of arachidonic acid. In this study we were interested in examining if the enzymatic system involved in the remodeling of membrane PC presented any selectivity for DHA in rat heart. The enzymes that were studied from sequential incubations carried out in parallel, were acyl-CoA synthetase (EC 6.2.1.3) and acyl-CoA:lysophosphatidylcholine acyltransferase (EC 2.3.1.23) (ACLAT). The heart preparations examined were homogenates of whole heart and of purified cultured rat ventricular myocytes.Results showed that ACLAT tended to preferentially incorporate into PC the polyunsaturated fatty acids of the n-6 series (+30%) rather than those of the n-3 series. DHA, however, inhibited the incorporation of arachidonic acid (AA) into PC by 50% at a molar ratio (DHA/AA) of 1.5. This phenomenon seems to be related to the competitive inhibition exerted by DHA on the thio-esterification of AA, a reaction catalyzed by acyl-CoA synthetase. This inhibitory effect appears to be dependent on the kinetic properties of the acyl-CoA synthetase toward DHA which, among the fatty acids examined, exhibited the lowest apparent Km and Vmax.It is suggested that the intracellular pool of DHA-CoA is the determinant species in altering the DHA composition of cardiac PC in animals given fish oils.