人红细胞膜和血浆游离脂肪酸成分分析及其对膜微粘度的影响

采用高效液相色谱（ＨＰＬＣ）和荧光偏振技术测定了４２例正常人红细胞膜和血浆游离脂肪酸（ＦＦＡ）及膜微粘度,并探讨了膜脂肪酸和血浆ＦＦＡ构成与膜微粘度之间的关系。结果表明：正常人红细胞膜主要由廿二碳六烯酸（Ｃ２２∶６）、花生四烯酸（Ｃ２０∶４）、亚油酸（Ｃ１８∶２）、软脂酸（Ｃ１６∶０）、油酸（Ｃ１８∶１）和硬脂酸（Ｃ１８∶０）等六种脂肪酸组成。血浆ＦＦＡ构成与膜脂肪酸相似,但不含Ｃ２２∶６而含十四烷酸（Ｃ１４∶０）。红细胞膜各脂肪酸含量大多与其血浆浓度呈明显正相关。红细胞膜微粘度与膜软脂酸和硬脂酸呈明显正相关,与膜廿二碳六烯酸和花生四烯酸呈明显负相关。提示红细胞膜脂肪酸组成受血浆ＦＦＡ成分影响;而红细胞膜脂肪酸成分对膜微粘度亦有重要影响     

热环境对大鼠纹状体神经元细胞膜的损伤作用

采用原代培养的大鼠纹状体神经元,施予43℃热环境处理1h。用气,质联用的方法测定细胞膜和细胞中的脂肪酸水平,主要是花生四烯酸的水平;荧光偏振法测细胞膜流动性,用[^3H]花生四烯酸大肠杆菌膜检测细胞内磷脂酶A2活性。发现细胞内大量存在的脂肪酸水平在热环境处理前后没有明显差别,而花生四烯酸的水平明显升高。热处理造成细胞膜的流动性明显降低,同时明显增加了神经元内磷脂酶A2的活性。表明热处理明显影响神经元细胞膜的流动性和磷脂代谢,进而影响细胞膜的功能,而热对细胞膜的损伤作用可能就是热致神经元损伤的重要事件。     

脂类物质抗菌的研究进展

研究发现人体内存在多种体内合成的抗菌物质,其中蛋白质类与糖类物质的研究报道较多。最近的研究发现脂类物质,包括饱和脂肪酸(月桂酸、棕榈酸)、单不饱和脂肪酸(棕榈烯酸、油酸)、多不饱和脂肪酸(二十碳五烯酸、二十二碳六烯酸、花生四烯酸)以及类固醇(维生素D、性类固醇)等也具有抗菌作用。脂类物质一方面可以通过调节多种炎性细胞和免疫细胞调动自身免疫系统发挥间接抗菌作用,另一方面也可以通过直接插入细菌细胞膜,影响其完整性发挥直接抗菌作用。本研究对各种脂类物质抗菌作用及其相关机制的进展作一综述。     

被孢霉生物合成花生四烯酸的初步研究

花生四烯酸不仅是细胞膜的重要成分,在维持细胞膜的结构与功能上发挥重要作用,而且是人体前列腺素合成的前体物质,对人体生理功能具有重要的调节作用。近年来研究发现,花生四烯酸在保护皮肤、降低胆固醇、抑制血小板聚集、提高免疫能力、促进胎儿发育等方面具有独特的...     

实时PCR分析△5脱饱和酶在花生四烯酸合成中的作用

花生四烯酸是人体的必需脂肪酸,具有独特的生物活性。 Δ5脱饱和酶是花生四烯酸生物合成途径中催化二高-γ-亚麻酸脱饱和为花生四烯酸的酶。本研究通过实时定量PCR技术,检测了Δ5脱饱和酶在不同花生四烯酸产量的高山被孢霉(Mortierella alpina)菌株M10,M6和M23中的mRNA表达水平,以及在高产花生四烯酸菌株M6培养过程中的mRNA表达水平的动态变化,发现Δ5脱饱和酶基因的mRNA表达水平与花生四烯酸的产生之间存在明显的线性关系,表明Δ5脱饱和酶是高山被孢霉中花生四烯酸合成途径中起到了非常重要的作用。     

花生四烯酸的生物活性及其钙信号转导作用

花生四烯酸（arachidonic acid,AA）以酯化形式在膜磷脂中,细胞兴奋时多种信号转导途径可引起游离AA释放,并迅速代谢为具有生物活性的炎症物质,参与细胞免疫和炎症反应。目前大量研究表明,AA本身还直接参与细胞内生物功能的调节,包括影响酶功能,调控各种离子通道,尤其是直接导致细胞内信号转导,成为细胞膜受体兴奋－细胞内生物反应偶联的第二信使。但AA的作用机制及其生理和病理生理意义有待进一步研究。     

大鼠壁细胞基底膜容积致敏感氯通道电流

为研究胃粘膜壁细胞容积致敏感氯通道电流的定位、电生理特征及药物效应并推断其在壁细胞病理生理过程中所起作用,对急性分离的大鼠胃粘膜壁细胞进行全细胞膜片钳记录,将电极液设置为高渗（Δ≈７０ｍＯｓｍ）,使细胞出现稳定的体积增大后,在其基底膜上记录到容积致敏感氯通道电流（ｖｏｌｕｍｅ－ｓｅｎｓｉｔｉｖｅｃｈｌｏｒｉｄｅｃｈａｎｎｅｌｃｕｒｅｎｔ,ＶＳＣｌＣＣ）,该电流具有外向整流性及电压和时间依赖性,可被１０μｍｏｌ／Ｌ花生四烯酸（ａｒａｃｈｉｄｏｎｉｃａｃｉｄ,ＡＡ）可逆性抑制（７１．３±１０．９％）。细胞外液ｐＨ值由７．４降低至４．０,ＶＳＣｌＣＣ的幅度显著下降（６３．１±１４．０％）,而其激活及失活动力学无改变;ｐＨ升高至９．０对ＶＳＣｌＣＣ无影响。壁细胞ＶＳＣｌＣＣ对细胞内外钙离子浓度均呈现明显的依赖性。结果表明,壁细胞基底膜上存在本底氯通道和容积致敏感氯通道两种不同的氯离子通道。ＶＳＣｌＣＣ可能参与某些胃粘膜疾病的病理生理过程。     

白细胞的花生四烯酸代谢

炎症时白细胞活动旺盛,细胞膜磷脂分解生成花生四烯酸,以此为原料,一方面生成PGs 和TX;另一方面生成HETE 和LT 等。这些物质吸引白细胞到炎灶,有的物质还可引起溶酶体酶释放以及微血管效应,影响炎症的发生和发展。除炎症外,这类物质和哮喘、心肌缺血、免疫、癌症等都有关。     

植物多糖抗肿瘤作用与机理研究：Ⅲ,茯苓多糖（PPS）和刺五加多糖（A…

我们先前的研究表明,植物多糖抑制体外培养的小鼠肉瘤Ｓ１８０细胞增殖并使细胞膜磷脂含量减少,同时抑制膜磷脂酰肌醇转换。为进一步探讨植物多糖与膜磷脂的关系,本文采用毛细管柱气相色谱法分析了茯苓多糖（ＰＰＳ）、刺五加多糖（ＡＳＰＳ）与Ｓ１８０细胞一同温育２４ｈ后,细胞膜磷脂和中性脂的脂肪酸组成变化,发现中性脂的脂肪酸组成和不饱和性不受影响,磷脂的脂肪酸组成发生明显改变,花生四烯酸（Ｃ２０：４）和豆蔻酸（     

高山被孢霉产花生四烯酸及其遗传改造的研究进展

花生四烯酸作为一种重要的多价不饱和脂肪酸,因其具有多种生理功能而被认为是潜在的食品添加剂和药物。近年来,利用高山被孢霉合成花生四烯酸已成为研究热点。前期相关研究主要集中在菌种选育及发酵调控方面。随着研究的不断深入,关于高山被孢霉合成花生四烯酸的代谢途径的研究取得了较大进展。以下简要概述前期工作进展,着重论述花生四烯酸合成途径的关键酶及其高山被孢霉的遗传改造的研究情况,包括生物合成花生四烯酸代谢途径、关键酶及其应用、高山被孢霉的遗传操作系统的构建以及遗传改造的应用,并对其研究前景进行了展望。     

ATP-sensitive K+ channels in insulinoma cells are activated by nonesterified fatty acids.

Both 86Rb+ efflux experiments and electrophysiological studies have shown that arachidonic acid and other nonesterified fatty acids activate ATP-sensitive K+ channels in insulinoma cells (HIT-T15). Activation was observed with arachidonic, oleic, linoleic, and docosahexaenoic acid but not with myristic, stearic, and elaidic acids. Fatty acid activation of ATP-sensitive K+ channels was blocked by antidiabetic sulfonylureas such as glibenclamide. The activating effect of arachidonic acid was unaltered by indomethacin and by nordihydroguaiaretic acid, indicating that it is not due to metabolites of arachidonic acid via cyclooxygenase or lipoxygenase pathways. Moreover, the nonmetabolizable analogue of arachidonic acid, eicosatetraynoic acid, was an equally potent activator. Activation of ATP-sensitive K+ channels by fatty acids was potentiated by diacylglycerol and was inhibited by calphostin C, an inhibitor of protein kinase C. These findings indicate that fatty acid activation of ATP-sensitive K+ channels is most likely due to the participation of arachidonic acid (and other fatty acid)-activated protein kinase C isoenzymes. Activation of ATP-sensitive K+ channels by nonesterified fatty acids is not involved in the control of insulin secretion since arachidonic acid stimulates insulin secretion from insulinoma cells instead of inhibiting it.     

Regulation of potassium channels in coronary smooth muscle by adenoviral expression of cytochrome P-450 epoxygenase

Epoxyeicosatrienoic acids (EETs) are endothelium-derived cytochrome P-450 (CYP) metabolites of arachidonic acid that relax vascular smooth muscle by large-conductance calcium-activated potassium (BK(Ca)) channel activation and membrane hyperpolarization. We hypothesized that if smooth muscle cells (SMCs) had the capacity to synthesize EETs, endogenous EET production would increase BK(Ca) channel activity. Bovine coronary SMCs were transduced with adenovirus coding the CYP Bacillus megaterium -3 (F87V) (CYP BM-3) epoxygenase that metabolizes arachidonic acid exclusively to 14(S),15(R)-EET. Adenovirus containing the cytomegalovirus promoter-Escherichia coli beta-galactosidase was used as a control. With the use of an anti-CYP BM-3 (F87V) antibody, a 124-kDa immunoreactive protein was detected only in CYP BM-3-transduced cells. Protein expression increased with increasing amounts of virus. When CYP BM-3-transduced cells were incubated with [14C]arachidonic acid, HPLC analysis detected 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) and 14,15-EET. The identity of 14,15-EET and 14,15-DHET was confirmed by mass spectrometry. In CYP BM-3-transduced cells, methacholine (10(-5) M) increased 14,15-EET release twofold and BK(Ca) channel activity fourfold in cell-attached patches. Methacholine-induced increases in BK(Ca) channel activity were blocked by the CYP inhibitor 17-octadecynoic acid (10(-5) M). 14(S),15(R)-EET was more potent than 14(R),15(S)-EET in relaxing bovine coronary arteries and activating BK(Ca) channels. Thus CYP BM-3 adenoviral transduction confers SMCs with epoxygenase activity. These cells acquire the capacity to respond to the vasodilator agonist by synthesizing 14(S),15(R)-EET from endogenous arachidonic acid to activate BK(Ca) channels. These studies indicate that 14(S),15(R)-EET is a sufficient endogenous activator of BK(Ca) channels in coronary SMCs.     

Arachidonate dilates basilar artery by lipoxygenase-dependent mechanism and activation of K(+) channels

Dilatation of cerebral arterioles in response to arachidonic acid is dependent on activity of cyclooxygenase. In this study, we examined mechanisms that mediate dilatation of the basilar artery in response to arachidonate. Diameter of the basilar artery (baseline diameter = 216 +/- 7 micrometer) (means +/- SE) was measured using a cranial window in anesthetized rats. Arachidonic acid (10 and 100 microM) produced concentration-dependent vasodilatation that was not inhibited by indomethacin (10 mg/kg iv) or N(G)-nitro-L-arginine (100 microM) but was inhibited markedly by baicalein (10 micrometerM) or nordihydroguaiaretic acid (NDGA; 10 microM), inhibitors of the lipoxygenase pathway. Dilatation of the basilar artery was also inhibited markedly by tetraethylammonium ion (TEA; 1 mM) or iberiotoxin (50 nM), inhibitors of calcium-dependent potassium channels. For example, 10 microM arachidonate dilated the basilar artery by 19 +/- 7 and 1 +/- 1% in the absence and presence of iberiotoxin, respectively. Measurements of membrane potential indicated that arachidonate produced hyperpolarization of the basilar artery that was blocked completely by TEA. Incubation with [(3)H]arachidonic acid followed by reverse-phase and chiral HPLC indicated that the basilar artery produces relatively small quantities of prostanoids but large quantities of 12(S)-hydroxyeicosatetraenoic acid (12-S-HETE), a lipoxygenase product. Moreover, the production of 12-HETE was inhibited by baicalein or NDGA. These findings suggest that dilatation of the basilar artery in response to arachidonate is mediated by a product(s) of the lipoxygenase pathway, with activation of calcium-dependent potassium channels and hyperpolarization of vascular muscle.     

Ca2+ selectivity and fatty acid specificity of the noncapacitative,arachidonate-regulated Ca2+ (ARC) channels

The arachidonate-regulated, Ca(2+)-selective ARC channels represent a novel receptor-activated pathway for the entry of Ca(2+) in nonexcitable cells that is entirely separate from the widely studied store-operated, Ca(2+) release-activated Ca(2+) channels. Activation of ARC channels occurs specifically at the low agonist concentrations typically associated with oscillatory Ca(2+) signals and appears to provide the predominant mode of Ca(2+) entry under these conditions (Mignen, O., Thompson, J. L., and Shuttleworth, T. J. (2001) J. Biol. Chem. 276, 35676-35683). In this study we demonstrate that ARC channels are present in a variety of different cell types including both cell lines and primary cells. Examination of their pharmacology revealed that currents through these channels are significantly inhibited by low concentrations (< 5 microm) of Gd(3+), are unaffected by 100 microm 2-aminoethyoxydiphenyl borane, and are not activated by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (100 microm). Their selectivity for Ca(2+) was assessed by determining the EC(50) for external Ca(2+) block of the monovalent currents observed in the absence of external divalent cations. The value obtained (150 nm) indicates that the Ca(2+) selectivity of ARC channels is extremely high. Examination of the ability of various fatty acids, including arachidonic acid, to activate the ARC channels demonstrated that activation does not reflect any nonspecific membrane fluidity or detergent effects, shows a high degree of specificity for arachidonic acid over other fatty acids (especially monounsaturated and saturated fatty acids), and is independent of any arachidonic acid metabolite. Moreover, studies using the charged analogue arachidonyl coenzyme A demonstrate that activation of the ARC channels reflects an action of the fatty acid specifically at the internal face of the plasma membrane. Whether this involves a direct action of arachidonic acid on the channel protein itself or an action on some intermediary molecule is, at present, unclear.     

Eicosanoids inhibit the G-protein-gated inwardly rectifying potassium channel (Kir3) at the Na+/PIP2 gating site

We previously showed that activation of the human endothelin A receptor (HETAR) by endothelin-1 (Et-1) selectively inhibits the response to mu opioid receptor (MOR) activation of the G-protein-gated inwardly rectifying potassium channel (Kir3). The Et-1 effect resulted from PLA2 production of an eicosanoid that inhibited Kir3. In this study, we show that Kir3 inhibition by eicosanoids is channel subunit-specific, and we identify the site within the channel required for arachidonic acid sensitivity. Activation of the G-protein-coupled MOR by the selective opioid agonist D-Ala(2)Glyol, enkephalin, released Gbetagamma that activated Kir3. The response to MOR activation was significantly inhibited by Et-1 activation of HETAR in homomeric channels composed of either Kir3.2 or Kir3.4. In contrast, homomeric channels of Kir3.1 were substantially less sensitive. Domain deletion and channel chimera studies suggested that the sites within the channel required for Et-1-induced inhibition were within the region responsible for channel gating. Mutation of a single amino acid in the homomeric Kir3.1 to produce Kir3.1(F137S)(N217D) dramatically increased the channel sensitivity to arachidonic acid and Et-1 treatment. Complementary mutation of the equivalent amino acid in Kir3.4 to produce Kir3.4(S143T)(D223N) significantly reduced the sensitivity of the channel to arachidonic acid- and Et-1-induced inhibition. The critical aspartate residue required for eicosanoid sensitivity is the same residue required for Na(+) regulation of PIP(2) gating. The results suggest a model of Kir3 gating that incorporates a series of regulatory steps, including Gbetagamma, PIP(2), Na(+), and arachidonic acid binding to the channel gating domain.     

GVI phospholipase A2 role in the stimulatory effect of sphingosine-1-phosphate on TRPC5 cationic channels

The Transient Receptor Potential Canonical 5 (TRPC5) protein forms calcium-permeable cationic channels that are stimulated by G protein-coupled receptor agonists. The signaling pathways of such agonist effects are poorly understood. Here we investigated the potential for involvement of lysophosphatidylcholine (LPC) and arachidonic acid generated by group 6 (GVI) phospholipase A2 (PLA2) enzymes, focusing on stimulation of TRPC5 by sphingosine-1-phosphate (S1P) which acts via a pertussis toxin-sensitive (Gi/o protein) pathway without Ca²⁺-release. Experiments were on HEK 293 cells containing conditional expression of human TRPC5. Channel activity was recorded using an intracellular calcium indicator or whole-cell patch-clamp and PLA2 activity was detected using ³H-arachidonic acid. S1P stimulated PLA2 and TRPC5 activities. Both effects were suppressed by the GVI PLA2 inhibitor bromoenol lactone. Knock-down of GVI PLA2 by RNA interference suppressed channel activity evoked by S1P whereas activity evoked by the direct channel stimulator LPC was unaffected. Arachidonic acid did not stimulate the channels. Prior exposure of channels to LPC but not arachidonic acid suppressed channel activity evoked by S1P but not gadolinium, a putative direct stimulator of the channels. The data suggest roles of LPC and GVI PLA2 in S1P-evoked TRPC5 activity.     

Arachidonic acid regulates surface expression of epithelial sodium channels

Epithelial Na+ channels (ENaCs) are regulated by the phospholipase A2 (PLA2) product arachidonic acid. Pharmacological inhibition of PLA2 with aristolochic acid induced a significant increase in amiloride-sensitive currents in Xenopus oocytes expressing ENaC. Arachidonic acid or 5,8,11,14-eicosatetraynoic acid (ETYA), a non-metabolized analog of arachidonic acid, induced a time-dependent inhibition of Na+ transport. These effects were also observed by co-expression of a calcium-independent or a calcium-dependent PLA2. Channels with a truncated alpha, beta,or gamma C terminus were not inhibited by arachidonic acid or ETYA. Furthermore, mutation of Tyr618 in the PY motif of the beta subunit abrogated the inhibitory effect of ETYA, suggesting that intact PY motifs participate in arachidonic acid-mediated ENaC inhibition. Analyses of channels expressing a series of beta subunit C-terminal truncations revealed a second region N-terminal to the PY motif (spanning residues betaVal580-betaGly599) that allowed for ETYA-mediated ENaC inhibition. Analyses of both ENaC surface expression and ENaC trafficking with mutants that either gate channels open or closed in response to [(2-(trimethylammonium) ethyl] methanethiosulfonate bromide, or with brefeldin A, suggest that ETYA reduces channel surface expression by inhibiting ENaC exocytosis and increasing ENaC endocytosis.     

Regulation of recombinant human brain tandem P domain K+ channels by hypoxia: a role for O2 in the control of neuronal excitability?

The tandem P domain potassium channels, TREK1 and TASK1, are expressed throughout the brain but expression patterns do not significantly overlap. Since normal pO₂ in central nervous tissue is as low as 20 mmHg and can decrease even further in ischemic disease, it is important that the behaviour of human brain ion channels is studied under conditions of acute and chronic hypoxia. This is especially true for brain-expressed tandem P-domain channels principally because they are important contributors to neuronal resting membrane potential and excitability. Here, we discuss some recent data derived from two recombinant tandem P-domain potassium channels, hTREK1 and hTASK1. Hypoxia represents a potent inhibitory influence on both channel types and occludes the activation by arachidonic acid, intracellular acidosis and membrane deformation of TREK1. This casts doubt on the idea that TREK1 activation during brain ischemia might facilitate neuroprotection via hyperpolarising neurons in which it is expressed. Interestingly, hypoxia is unable to regulate alkalotic inhibition of TREK1 suggesting that this channel may be more intimately involved in control of excitability during physiological or pathological alkalosis.     

Connexin hemichannels and gap junction channels are differentially influenced by lipopolysaccharide and basic fibroblast growth factor

Gap junction (GJ) channels are formed by two hemichannels (connexons), each contributed by the cells taking part in this direct cell-cell communication conduit. Hemichannels that do not interact with their counterparts on neighboring cells feature as a release pathway for small paracrine messengers such as nucleotides, glutamate, and prostaglandins. Connexins are phosphorylated by various kinases, and we compared the effect of various kinase-activating stimuli on GJ channels and hemichannels. Using peptides identical to a short connexin (Cx) amino acid sequence to specifically block hemichannels, we found that protein kinase C, Src, and lysophosphatidic acid (LPA) inhibited GJs and hemichannel-mediated ATP release in Cx43-expressing C6 glioma cells (C6-Cx43). Lipopolysaccharide (LPS) and basic fibroblast growth factor (bFGF) inhibited GJs, but they stimulated ATP release via hemichannels in C6-Cx43. LPS and bFGF inhibited hemichannel-mediated ATP release in HeLa-Cx43 cells, but they stimulated it in HeLa-Cx43 with a truncated carboxy-terminal (CT) domain or in HeLa-Cx26, which has a very short CT. Hemichannel potentiation by LPS was inhibited by blockers of the arachidonic acid metabolism, and arachidonic acid had a potentiating effect like LPS and bFGF. We conclude that GJ channels and hemichannels display similar or oppositely directed responses to modulatory influences, depending on the balance between kinase activity and the activity of the arachidonic acid pathway. Distinctive hemichannel responses to pathological stimulation with LPS or bFGF may serve to optimize the cell response, directed at strictly controlling cellular ATP release, switching from direct GJ communication to indirect paracrine signaling, or maximizing cell-protective strategies.     

Coupled potassium channels induced by arachidonic acid in cultured neurons

Exposure of the inside surface of patches of membrane excised from cultured rat hippocampal neurons to arachidonic acid (10-100 microM) caused the appearance of potassium currents of variable amplitude similar to those activated by GABA or baclofen in cell-attached patches. The amplitude of single-channel currents increased with time after exposure to 20 or 50 microM arachidonic acid and also increased when arachidonic acid concentration was increased from 20 to 50 or 100 microM. Current-amplitude probability histograms had peaks at integral multiples of an 'elementary' current. It is proposed that arachidonic acid or its metabolites cause synchronous opening and closing of coupled conducting units (co-channels) in cell membranes.