Arachidonic acid induces a prolonged inhibition of glutamate uptake into glial cells

Activation of NMDA (N-methyl-D-aspartate) receptors by neurotransmitter glutamate stimulates phospholipase A2 to release arachidonic acid. This second messenger facilitates long-term potentiation of glutamatergic synapses in the hippocampus, possibly by blocking glutamate uptake. We have studied the effect of arachidonic acid on glutamate uptake into glial cells using the whole-cell patch-clamp technique to monitor the uptake electrically. Micromolar levels of arachidonic acid inhibit glutamate uptake, mainly by reducing the maximum uptake rate with only small effects on the affinity for external glutamate and sodium. On removal of arachidonic acid a rapid (5 minutes) phase of partial recovery is followed by a maintained suppression of uptake lasting at least 20 minutes. Surprisingly, the action of arachidonic acid is unaffected by cyclo-oxygenase or lipoxygenase inhibitors suggesting that it inhibits uptake directly, possibly by increasing membrane fluidity. As blockade of phospholipase A2 prevents the induction of long-term potentiation (LTP), inhibition of glutamate uptake by arachidonic acid may contribute to the increase of synaptic gain that occurs in LTP. During anoxia, release of arachidonic acid could severely compromise glutamate uptake and thus contribute to neuronal death.     

Leukotriene B, a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes

Arachidonic acid is metabolised either by cyclooxygenases to produce prostaglandins and thromboxanes or by lipoxygenases to produce mono-, di- and trihydroxyeicosatetraenoic acids (HETEs). Polymorphonuclear leukocytes (PMNs) release HETEs, including mono- and dihydroxy fatty acids, when exposed to stimuli such as the calcium ionophore A23187 (refs 1, 2). The mono-HETEs are assumed to be of particular importance with respect to effects on leukocyte function because they have been shown to possess both chemotactic and chemokinetic activities towards PMNs and eosinophils. However, we have now shown that the chemokinetic and aggregating activities released from rat and human PMNs exposed to ionophore A23187 (ref. 5) are not due to the release of mono-HETEs but to that of 5, 12-di-HETE (leukotriene B). This compound is active over the concentration range 10 pg ml-1 to 5 ng ml-1.     

Enzymatic assembly of slow reacting substance

When basophils or mast cells are stimulated by a specific antigen they release chemical mediators, including a potent bronchoconstrictor, slow reacting substance of anaphylaxis (SRS-A). The structure of SRS from a mouse mastocytoma and rat basophilic leukaemia (RBL-1) cells has been identified as a thioether or arachidonic acid and glutathione [not a thioether of cystene as was originally thought]. SRS has been named leukotriene (LT) C and may be formed by a novel lipoxygenase pathway which also synthesizes 5,6-oxido-7,9,11,14-icosatetraenoic acid (LTA) and 5,12-dihydroxy-6,8,10,14-icosatetraenoic acid (LTB). Homogenates of RBL-1 cells, when incubated with C-arachidonic acid, form 5-hydroxy-icosatetraenoic acid (5-HETE) and 5,12-dihydroxy- and 5,6-dihydroxy-icosatetraenoic acid. The latter is the spontaneous breakdown product of the labile intermediate LTA. Formation of both compounds is stimulated by calcium. We have now produced biologically active SRS in a cell-free system generated from RBL-1 cells. Glutathione was essential for SRS synthesis and calcium stimulated its formation.     

在金刚石膜上电化学氧化对硝基苯酚

利用金刚石膜电极电化学氧化技术可以消除环境污染,该技术可以有效地处理含苯酚、氯酚或其他含酚的化合物以及羧基酸,在大多数的情况下,这些有机物可以被完全矿化.由此表明,电化学氧化技术处理污水中含有苯环的有机物是很有前景的,但相关报道很少.含硝基的有机化合物是工业和农     

Lipoxygenase metabolites of arachidonic acid as second messengers for presynaptic inhibition of Aplysia sensory cells

Biochemical and biophysical studies on Aplysia sensory neurons indicate that inhibitory responses to the molluscan peptide FMRFamide are mediated by lipoxygenase metabolites of arachidonic acid. These compounds are a new class of second messengers in neurons.     

Regulation of the arachidonic acid-stimulated respiratory burst in neutrophils by intracellular and extracellular calcium

The respiratory burst is an important physiological function of the neutrophils in killing the bacteria invading in human body. We used chemiluminescence method to measure the exogenous arachidonic acid-stimulated respiratory burst, and measured the cytosolic free calcium concentration in neutrophils by the fluorescence method. It was found that, on one hand, the arachidonic acid-stimulated respiratory burst was enhanced by elevating the cytosolic free calcium concentration in neutrophils with a potent endomembrane Ca2+-ATPase inhibitor, Thapsgargin; on the other hand, chelating the intracellular or extracellular calcium by EGTA or BAPTA inhibited the respiratory burst. Results showed that calcium plays an important regulatory role in the signaling pathway involved in the exogenous arachidonic acid-stimulated respiratory burst of neutrophils.     

G-protein beta gamma-subunits activate the cardiac muscarinic K+-channel via phospholipase A2

Muscarinic receptors of cardiac pacemaker and atrial cells are linked to a potassium channel (IK.ACh) by a pertussis toxin-sensitive GTP-binding protein. The dissociation of G-proteins leads to the generation of two potential transducing elements, alpha-GTP and beta gamma. IK.ACh is activated by G-protein alpha- and beta gamma-subunits applied to the intracellular surface of inside-out patches of membrane. beta gamma has been shown to activate the membrane-bound enzyme phospholipase A2 in retinal rods. Arachidonic acid, which is produced from the action of phospholipase A2 on phospholipids, is metabolized to compounds which may act as second messengers regulating ion channels in Aplysia. Muscarinic receptor activation leads to the generation of arachidonic acid in some cell lines. We therefore tested the hypothesis that beta gamma activates IK.ACh by stimulation of phospholipase A2. When patches were first incubated with antibody that blocks phospholipase A2 activity, or with the lipoxygenase inhibitor, nordihydroguaiaretic acid, beta gamma failed to activate IK.ACh. Arachidonic acid and several of its metabolites derived from the 5-lipoxygenase pathway, activated the channel. Blockade of the cyclooxygenase pathway did not inhibit arachidonic acid-induced channel activation. We conclude that the beta gamma-subunit of G-proteins activates IK.ACh by stimulating the production of lipoxygenase-derived second messengers.     

Arachidonic acid metabolites as intracellular modulators of the G protein-gated cardiac K+ channel

Arachidonic acid is released from cell membranes in response to receptor-dependent as well as receptor-independent stimulation in various cells, including cardiac myocytes. Arachidonic acid is converted to prostaglandins by cyclooxygenase and to leukotrienes by 5-lipoxygenase, metabolites which are very biologically active and modulate cellular functions such as platelet aggregation, smooth muscle contraction and neural excitation. The molecular mechanisms underlying their modulations are, however, still badly understood. Here, we report that the 5-lipoxygenase metabolites of arachidonic acid activate the pertussis toxin-sensitive G protein-gated muscarinic K+ channel (IK.ACh): arachidonic acid activation of IK.ACh was prevented by the lipoxygenase inhibitors, nordihydroguaiaretic acid and AA-861; leukotriene A4 and C4 activated IK.ACh. The activation occurred in pertussis toxin-treated atrial cells and ceased when inside-out patches were formed but the patches were still susceptible to stimulation by GTP and to inhibition by GDP-beta-S. These results indicate that arachidonic acid metabolites may stimulate the G-protein in a receptor-independent way.     

Arachidonic acid metabolites as mediators of somatostatin-induced increase of neuronal M-current

The M-current (IM) is a time- and voltage-dependent K+ current that persists at slightly depolarized membrane potentials. IM is reduced by muscarinic cholinergic agonists and certain peptides, and is thought to be responsible in part for the slow and late slow excitatory postsynaptic potentials in sympathetic neurons. Recently, we reported that IM in hippocampal neurons was also augmented by somatostatin-14 and -28 suggesting that two different receptors reciprocally regulate one neuronal channel type. Muscarinic effects on IM may be mediated by various components of the phosphatidylinositol phosphate pathway. We now report the involvement of a different second messenger pathway, that generated by phospholipase A2, in the somatostatin-induced augmentation of IM in hippocampal cells. This pathway generates arachidonic acid from which leukotrienes can be produced by lipoxygenases. We find that the IM-augmenting effects of somatostatin are abolished by two substances that can inhibit phospholipase A2, quinacrine and 4-bromophenacyl bromide, and that both arachidonic acid and leukotriene C4 mimic the effects of somatostatin-14 on hippocampal pyramidal neurons in vitro. Arachidonic and somatostatin effects are blocked by a lipoxygenase inhibitor, implicating an arachidonic acid metabolite, perhaps a leukotriene, in the somatostatin effect.     

Potentiation of NMDA receptor currents by arachidonic acid.

Arachidonic acid is released by phospholipase A2 when activation of N-methyl-D-aspartate (NMDA) receptors by neurotransmitter glutamate raises the calcium concentration in neurons, for example during the initiation of long-term potentiation and during brain anoxia. Here we investigate the effect of arachidonic acid on glutamate-gated ion channels by whole-cell clamping isolated cerebellar granule cells. Arachidonic acid potentiates, and makes more transient, the current through NMDA receptor channels, and slightly reduces the current through non-NMDA receptor channels. Potentiation of the NMDA receptor current results from an increase in channel open probability, with no change in open channel current. We observe potentiation even with saturating levels of agonist at the glutamate- and glycine-binding sites on these channels; it does not result from conversion of arachidonic acid to lipoxygenase or cyclooxygenase derivatives, or from activation of protein kinase C. Arachidonic acid may act by binding to a site on the NMDA receptor, or by modifying the receptor's lipid environment. Our results suggest that arachidonic acid released by activation of NMDA (or other) receptors will potentiate NMDA receptor currents, and thus amplify increases in intracellular calcium concentration caused by glutamate. This may explain why inhibition of phospholipase A2 blocks the induction of long-term potentiation.     

白玉蜗牛肉的脂肪酸组成特点分析

利用索氏提取法提取白玉蜗牛肉冻干粉中的脂肪酸,通过三氟化硼法进行甲酯化。以气相色谱-质谱联用仪进行组分鉴定。结果表明,蜗牛肉冻干粉的总脂得率为2.73 ％,在总脂的甲酯化产物中检测鉴定出15种脂肪酸,含9种不饱和脂肪酸,占总量的72.22 %,其中亚油酸占17.42 %,油酸占16.54 %,花生四烯酸占13.13 %,EPA占2.14 %。白玉蜗牛肉富含多不饱和脂肪酸(占总脂的48.03 %),可以用于多不饱和脂肪酸类新型功能食品与保健品的开发。     

Long-term heterosynaptic inhibition in Aplysia

Synaptic transmission between mechanosensory and motor neurons of the gill withdrawal reflex in Aplysia can undergo both short-term and long-term modulation. One form of short-term synaptic depression lasting minutes can be evoked by the peptide Phe-Met-Arg-Phe-amide (FMRFamide), and is mediated by the lipoxygenase pathway of arachidonic acid. We report here using cell culture, that the same monosynaptic sensory-to-motor component of the gill withdrawal reflex can also undergo long-term synaptic depression lasting 24 h after five applications of FMRFamide over a 2-h period. The long-term depression evoked by FMRFamide is transmitter-specific. Dopamine or low-frequency stimulation of sensory neurons, which also produce short-lasting synaptic depression in vivo, failed to evoke a long-term change. As is the case for long-term presynaptic facilitation of this connection with serotonin, the long-term depression, but not the short-term, can be blocked when applications of FMRFamide are given in the presence of anisomycin, a reversible inhibitor of protein synthesis. Thus, heterosynaptic depression parallels heterosynaptic facilitation in having a long-term as well as a short-term form, and in both cases the long-term modulation requires the synthesis of gene products not essential for the short-term changes.     

Arachidonic acid induces a long-term activity-dependent enhancement of synaptic transmission in the hippocampus

Long-term potentiation (LTP) is a widely studied model of the synaptic basis of information storage in the mammalian brain. The induction of LTP is triggered by the postsynaptic entry of calcium through the channel associated with the N-methyl-D-aspartate (NMDA) receptor, whereas its maintenance is mediated, at least in part, by presynaptic mechanisms. To explain how postsynaptic events can lead to an increase in transmitter release, we have postulated the existence of a retrograde messenger to carry information from the postsynaptic side of the synapse to recently active presynaptic terminals. Candidates for a retrograde messenger include arachidonic acid or one of its lipoxygenase metabolites. Here we report that weak activation of the perforant path, when given in the presence of arachidonic acid, leads to a slow-onset persistent increase in synaptic efficacy both in vivo and in vitro. The activity-dependent potentiation thus produced is accompanied by an increase in the release of glutamate, and is non-additive with tetanus-induced LTP. These observations indicate a role for arachidonic acid as a retrograde messenger in the later, but not the initial, stages of LTP.     

脂氧合酶--植物抗胁迫响应的关键酶

在植物抗胁迫响应中。脂氧合酶途径在抗性机制中非常重要．脂氧合酶是一种非血红素铁蛋白,在生物体内的主要作用是催化含有顺,顺-1,4-戊二烯结构的多不饱和脂肪酸加合氧分子,生成具有共轭双键的多不饱和脂肪酸的过氧化物及其一系列次生产物,如C3-,C6-,C9-和C12-醛、酮、醇、酸、酯等低分子量易挥发产物。它们和茉莉酸（茉莉酸甲酯）等构成植物抗性系统的信号分子诱导抗性蛋白合成及一系列抗胁迫反应．本文详述了脂氧合酶的结构、特性、作用机理、生理作用、活性测定方法和人工模拟。     

Enzymic activation and transfer of fatty acids as acyl-adenylates in mycobacteria

The metabolic repertoire in nature is augmented by generating hybrid metabolites from a limited set of gene products. In mycobacteria, several unique complex lipids are produced by the combined action of fatty acid synthases and polyketide synthases (PKSs), although it is not clear how the covalently sequestered biosynthetic intermediates are transferred from one enzymatic complex to another. Here we show that some of the 36 annotated fadD genes, located adjacent to the PKS genes in the Mycobacterium tuberculosis genome, constitute a new class of long-chain fatty acyl-AMP ligases (FAALs). These proteins activate long-chain fatty acids as acyl-adenylates, which are then transferred to the multifunctional PKSs for further chain extension. This mode of activation and transfer of fatty acids is contrary to the previously described universal mechanism involving the formation of acyl-coenzyme A thioesters. Similar mechanisms may operate in the biosynthesis of other lipid-containing metabolites and could have implications in engineering novel hybrid products.     

A primary determinant for lipoxygenase positional specificity

The three mammalian lipoxygenases are named according to the carbon position (5, 12 or 15) at which they catalyse the oxygenation of arachidonic acid; they are implicated in inflammatory disorders, for example 15-lipoxygenase is induced in atherosclerosis and can oxidize low-density lipoprotein to its atherogenic form. To identify what determines this positional specificity, we have exchanged conserved differences in the isoforms of 12- and 15-lipoxygenases. Substitution of methionine with valine at position 418 of human 15-lipoxygenase results in an enzyme that performs 12- and 15-lipoxygenation equally. This effect can be mimicked by incubating wild-type 15-lipoxygenase with a synthetically altered substrate which has its doubly allylic methylene carbons shifted by one carbon relative to arachidonic acid. Other mutations at the neighbouring amino acids 416 and 417 give an enzyme which performs 12- and 15-lipoxygenation in a ratio of 15:1. These results indicate that this region might position the substrate in the active site.     

蛇油中脂肪酸成分的GC—MS研究

用溶剂热抽提蛇油脂肪和ＫＯＨ／甲醇酯交换甲酯化,以气相色谱－质谱联用技术测定脂肪酸组成,鉴定了２９种脂肪酸成分,其中含量较高的成分有肉豆蔻酸,棕榈油酸,棕榈酸,亚油酸,油酸,硬脂酸,花生四烯酸,二十碳三烯酸,二十碳二烯酸,二十二碳四烯酸等．蛇油中不饱和脂肪酸的含量占脂肪酸总量的６３％以上．     

Leukotrienes are potent constrictors of human bronchi

Slow reacting substance of anaphylaxis (SRS-A) is released by various stimuli, including immunological challenge, and has long been considered an important mediator of immediate hypersensitivity reactions, such as bronchoconstriction in allergic asthma. Recently, slow reacting substances from several tissues have been identified and characterized as members of a newly discovered group of substances, the leukotrienes. Leukotrienes are generated from arachidonic acid and other polyunsaturated fatty acids in a pathway initially involving a lipoxygenase-catalysed oxygenation at C-5 (Fig. 1). This differs from the synthesis of prostaglandins and thromboxanes, where the initial transformation of arachidonic acid is catalysed by a cyclo oxygenase (Fig. 1). Recently, leukotriene C4(LTC4:5(S)-hydroxy,6(R)-S-glutathionyl-7,9-trans, 11,14-cis-eicosatetraenoic acid) and D4(LTD4:5(S)-hydroxy,6(R)-S-cysteinyl-glycyl-7,9-trans,11,14-cis-eicosatetraenoic acid) were found to have biological effects in several bioassay systems, which are strikingly similar to those previously reported for impure extracts of SRS-A. Here we report the remarkable contractile activity of both LTC4 and LTD4 on isolated human bronchi, which further emphasizes the possibility that leukotrienes are potent mediators of bronchoconstriction in man.     

Effects of L-lactic acid and D,L-lactic acid on viability and osteogenic differentiation of mesenchymal stem cells

Poly(lactic acid) (PLA) and other aliphatic polyesters containing the unit of lactic acid are very popular biodegradable materials. While the degradation products, lactic acids, have been worried to bring with negative influence on biocompatibility, the focused experimental studies are less reported. This study is aimed at an in vitro examination of cytotoxicity of both L-lactic acid and D,L-lactic acid. Mesenchymal stem cells (MSCs) derived from rat bone marrow are employed to test the cytotoxicity of the lactic acids. Considering that the addition of lactic acids not only introduces lactate groups but also alters medium pH and ion strength, these three candidate effects are examined in a decoupled way by setting different comparison groups. The results confirm that the change of medium pH is the predominant factor. It has also been found that D-lactate is more cytotoxic than L-lactate at high concentrations. Yet, either L-or D,L-lactic acids seem acceptable in most of medical applications, because the cytotoxicity is significant only when the concentrations are as high as 20 mmol/L for both of them.