Arachidonic Acid Methyl Ester Research Articles

Evaluation of Fatty Acid Contents and Biological Activities of Jurinea turcica.

The fatty acid profile, antioxidant/antibacterial, and cytotoxic effects of the extracts obtained from Jurinea turcica B.Doğan& A.Duran have been evaluated for the first time in the current study. The fatty acid profile of ethanolic extracts was determined using the Soxhlet extractor by a gas chromatography-mass spectrometer. The antioxidant and antibacterial activities were measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and ferrous reduction tests and the disc diffusion technique. Additionally, the cytotoxicity and wound healing assays were performed on A549 cells. The highest amount of component in the leaf extract was docosanoic acid methyl ester, whereas abundant arachidonic acid methyl ester was mainly found in the flower extract. The IC50 values, the 50 % scavenging value for the DPPH radical, were 179.13 and 124.67 μg/mL for the leaf and flower extracts, respectively. IC50 values (the half-maximal inhibitory concentration) were 10.4 and 24.7 μg/mL for the flower and leaf extracts, respectively. The leaf extract showed more potent antibacterial activity on Enterococcus faecalis (17 mm) and Staphylococcus aureus (16 mm) bacteria than the flower extract. In conclusion, the extracts of J. turcica have anti-cancerogenic and antibacterial effects. Leaf extracts have antibacterial and anti-metastatic effects, while flower extracts show antioxidant, cytotoxic, and apoptotic properties in A549 cells.

Characterization of natural rubber concerning its components and molecular weight in Taraxacum kok-saghyz Rodin using pyrolysis gas chromatography-mass spectrometry.

Taraxacum kok-saghyz Rodin (TKS) has abundant natural rubber in its root and the molecular weight of its natural rubber is higher than that in Hevea brasiliensis. Thus, TKS is an excellent alternative for the commercial production of natural rubber. The content and molecular weight of natural rubber are two qualitative indicators. Efficient determination for both indicators is still a challenge. In this study, we developed a method to simultaneously determine the content and molecular weight of natural rubber in TKS with pyrolysis－gas chromatography-mass spectrometry. The content of natural rubber was quantified by internal standard method. We optimized the pyrolysis temperature and chromatographic method during content determination. The limits of detection and quantification were 0.47 and 1.56μg, respectively. In addition, the arachidonic acid methyl ester, an unsaturated fatty acid proposed from the α-end group of natural rubber, was quantified to obtain the number of natural rubber polymers. Based on the content and the polymer number, we also quantified the molecular weight of natural rubber. Thus, the content and molecular weight of natural rubber were simultaneously determined in TKS. Our study provides a new perspective for the high throughput analysis of natural rubber.

Serum Metabonomics Reveals Key Metabolites in Different Types of Childhood Short Stature.

Nowadays, short stature (SS) in childhood is a common condition encountered by pediatricians, with an increase in not just a few families. Various studies related to the variations in key metabolites and their biological mechanisms that lead to SS have increased our understanding of the pathophysiology of the disease. However, little is known about the role of metabolite variation in different types of childhood SS that influence these biological processes and whether the understanding of the key metabolites from different types of childhood SS would predict the disease progression better. We performed a systematic investigation using the metabonomics method and studied the correlation between the three groups, namely, the control, idiopathic short stature (ISS), and short stature due to growth hormone deficiency (GHD). We observed that three pathways (viz., purine metabolism, sphingolipid signaling pathway, and sphingolipid metabolism) were significantly enriched in childhood SS. Moreover, we reported that two short peptides (Thr Val Leu Thr Ser and Trp Ile Lys) might play a significant role in childhood SS. Various metabolites in different pathways including 9,10-DiHOME, 12-HETE, 12(13)-EpOME, arachidonic acid methyl ester, glycerophospho-N-arachidonoyl ethanolamine, curvulinic acid (2-acetyl-3,5-dihydroxyphenyl acetic acid), nonanoic acid, and N'-(2,4-dimethylphenyl)-N-methylformamidine in human serum were compared between 60 children diagnosed with SS and 30 normal-height children. More investigations in this area may provide insights and enhance the personalized treatment approaches in clinical practice for SS by elucidating pathophysiology mechanisms of experimental verification.

Phytochemical Compounds of Branches from P. halepensis Oily Liquid Extract and S. terebinthifolius Essential Oil and Their Potential Antifungal Activity

In the present study, the antifungal activity of wood treated with Pinus halepensis branch n-hexane oily liquid extract (OLE) and Schinus terebinthifolius branch essential oil (EO) was evaluated against the growth of four phytopathogenic fungi—Bipolaris oryzae, Fusarium oxysporum, Fusarium solani, and Rhizoctonia solani. Air-dried wood samples of Pinus roxburghii were autoclaved, and each wood received 100 µL of the concentrated oils from P. halepensis and S. terebinthifolius. The main compounds identified in S. terebinthifolius branch EO were terpinen-4-ol (18.25%), cis-β-terpineol (15.60%), γ-terpinene (12.46%), sabinene (9.83%), α-terpinene (8.56%), and 4-thujanol (6.71%), while the main compounds in P. halepensis branch HeO were 2-undecenal (22.25%), 4-hydroxy-10-methyl-3,4,7,8,9,10-hexahydro-2H-oxecin-2-one (8.43%), (Z)-2-decenal (6.88%), nonanal (5.85%), (2E)-2-decenal (4.65%), (E,E)-2,4-decadienal (4.41%), arachidonic acid methyl ester (4.36%), and 2-(7-heptadecynyloxy)tetrahydro-2H-pyran (4.22%). P. halepensis OLE at a concentration of 3% showed the highest inhibition percentage of fungal growth (IPFG) of B. oryzae, followed by S. terebinthifolius EO at 3% and 2%, with IPFG values of 80%, 74.44%, and 71.66%, respectively. At a concentration of 3%, branch oils from S. terebinthifolius and P. halepensis were found to have the highest IPFG values with 45.55% and 40.55%, respectively, against F. oxysporum growth. Moderate to weak activity was found against F. solani when S. terebinthifolius EO and P. halepensis OLE were applied to wood. EO and OLE-treated wood samples at 3% produced inhibitions of 54.44% and 41.11%, respectively, against R. solani.

Polyunsaturated Fatty Acid Regulation of the Acid-Sensing Ion Channels

The Acid Sensing Ion Channels (ASICS) are proton activated, voltage-insensitive, sodium channels found throughout the peripheral and central nervous systems. Tissue acidosis can occur due to injury, ischemia, and inflammation which can lead to ASIC activation. Pain that occurs with several of these conditions, including ischemia and inflammation, has previously been linked to the activation of ASICs. In addition, ASICs have been shown to be able to sense several pro-inflammatory agents that are increased under these pathophysiological conditions. The polyunsaturated fatty acid (PUFA), Arachidonic Acid, is one such molecule that potentiates ASIC isoforms 1a, 2 and 3 at concentrations reached during inflammation. Interestingly, several other PUFAs have been shown to similarly potentiate ASIC1a. However, no study has yet to elucidate the mechanism of PUFA potentiation, nor has there been a thorough investigation of the how different properties of PUFAs modulate their effect on ASICs. Here we characterized how PUFA chain length, number of double bonds, location of double bonds and head group relate to potentiation of several ASIC isoforms. First, we demonstrate that PUFAs potentiates ASICs via an alkaline shift in the pH dependence of activation. In contrast to a previous finding, we show that the head group is critical for ASIC regulation by PUFAs. PUFAs with a negatively charged head group, like Arachidonoyl Glycine, dramatically shift the pH dependence of ASIC activation while uncharged head groups like Arachidonic Acid methyl ester have no effect on channel function. In addition, we show that the position of double bonds in the fatty acid tail are important for ASIC regulation as well. Finally, mutagenesis is used to localize possible PUFA interaction sites with ASICs.

Dietary seaweed (Ulva sp.) does not alter fatty acid profiles and concentration in South African juvenile dusky kob (Argyrosomus japonicus, Sciaenidae) fillet

ABSTRACT Seaweeds are potential feed additives for aquaculture but their effect on fish fatty acid content and profile is largely unknown. This study was, therefore, designed to assess the effect of incorporating green macroalgae seaweed (Ulva sp.) into diets of juvenile dusky kob (Argyrosomus japonicus) on fillet fatty acid profile and concentration. Five experimental diets were formulated to contain seaweed at the following inclusion levels: 0 (Ulva0), 50 (Ulva50), 100 (Ulva100), 150 (Ulva150) and 200 (Ulva200) g/kg commercial kob feed on a dry matter basis. Seventy-seven fingerlings (9.14 ± 0.30 g) were distributed into each of 20 experimental tanks and diets were randomly allocated to tanks such that each diet had 4 replicate tanks holding 77 fingerlings. There was no dietary effect on fillet fatty acids (FA) concentrations (P > .05). The concentration of C18:3n3 (α-linolenic acid) tended to increase with Ulva inclusion levels (P > .05). However, the opposite was observed with the fillet concentrations of both C20:4n6 (arachidonic acid methyl ester) and C20:5n3 (eicosapentaenoic acid (EPA)), but the decrease was also not significant. In conclusion, while Ulva-containing diets did not enhance the FA concentration in dusky kob fillet, the values were similar to those observed in fish fed on the relatively expensive commercial kob diet.

Nutritional Composition and Fatty Acids Analysis of <i>Capparis decidua</i> L. Fruits

The aim of this study was to find out the nutritional value, fatty acids and mineral composition of fruits of Capparis decidua L. This has been carried out by analyzing chemical composition for samples of the plant collected from Goz Abu kelab, Algutaina Road, White Nile State, Sudan. The proximate analysis and fatty acids composition were determined by analyzing sample of identified plants using different methods. The results of analysis showed that the fruits of C. decidua. contain total ash (6.34±0.06%), moisture (5.18±0.01%), total oil percentage (6.02±0.02%), crude fiber (6.19±0.5%), crude protein (14.32±0.2%), total carbohydrates (61.95±0.03%) and total sugar (0.42±0.1%). The fixed oil extracted from fruits were evaluated for chemical composition. The GC-FID analysis showed the presence of various saturated and unsaturated fatty acids such as Lauric acid (0.2080%) methyl ester, Tri decanoic acid methyl ester (0.3035%), Myristic acid methyl ester (0.5851%), palmatic acid methyl ester (46.0220%), Lamda-Linolenic acid methyl ester (0.6700%), Linolelaidic acid methyl ester (45.7910%), Cis- 11-Eicosenoic acid methyl ester (3.9941%), Arachidonic acid methyl ester (0.3080%), Erucic acid methyl ester (1.6876%) and Tricosanoic acid methyl ester (0.4307%). Fruits of C.decidua contained iron (81.8 mg/100g), sodium (26.9 mg/100g), potassium (2969.6 mg/100g), calcium (14.1 mg/100g) and manganese was not detected but it might been found in trace undetectable amounts. Generally, proximate compositions revealed that C.decidua fruits have better nutritional value as food and livestock feedstuff.

An efficient method for the enrichment of the arachidonic acid methyl ester from Mortierella alpina-derived crude oils

An improved method was developed for enriching arachidonic acid (AA) methyl ester from microbial oil by two-step low-temperature wet fractionation. The effects of solvent, operating temperature, and solvent-to-fatty acid methyl esters (FAMEs) ratio on the enrichment of AA were investigated. The best results were achieved when n-hexane was used as solvent. With operating temperatures in the range −30°C to −80°C and a FAMEs-to-solvent ratio of 1:5 (v/v), the proportion of AA methyl ester isolated could be increased to 83.76±2.78% with a yield of 52.89%. The total recovery of AA methyl ester would be further increased to 90.84% by recrystallization of the solid phases. The 20C, 22C saturated fatty acids were enriched by n-hexane or petroleum ether at −30°C, with concentrations increased 7.5-fold or 7.2-fold compared with their original levels, respectively. In addition, a method that combined alkali and acid catalysis of the transmethylation was the most conducive to the preparation of polyunsaturated fatty acid methyl esters.

Biosynthesis, isolation, and NMR analysis of leukotriene A epoxides: substrate chirality as a determinant of the cis or trans epoxide configuration

Leukotriene (LT)A₄ and closely related allylic epoxides are pivotal intermediates in lipoxygenase (LOX) pathways to bioactive lipid mediators that include the leukotrienes, lipoxins, eoxins, resolvins, and protectins. Although the structure and stereochemistry of the 5-LOX product LTA₄ is established through comparison to synthetic standards, this is the exception, and none of these highly unstable epoxides has been analyzed in detail from enzymatic synthesis. Understanding of the mechanistic basis of the cis or trans epoxide configuration is also limited. To address these issues, we developed methods involving biphasic reaction conditions for the LOX-catalyzed synthesis of LTA epoxides in quantities sufficient for NMR analysis. As proof of concept, human 15-LOX-1 was shown to convert 15S-hydroperoxy-eicosatetraenoic acid (15S-HPETE) to the LTA analog 14S,15S-trans-epoxy-eicosa-5Z,8Z,10E,12E-tetraenoate, confirming the proposed structure of eoxin A₄. Using this methodology we then showed that recombinant Arabidopsis AtLOX1, an arachidonate 5-LOX, converts 5S-HPETE to the trans epoxide LTA₄ and converts 5R-HPETE to the cis epoxide 5-epi-LTA₄, establishing substrate chirality as a determinant of the cis or trans epoxide configuration. The results are reconciled with a mechanism based on a dual role of the LOX nonheme iron in LTA epoxide biosynthesis, providing a rational basis for understanding the stereochemistry of LTA epoxide intermediates in LOX-catalyzed transformations.

Identification and absolute configuration of dihydroxy-arachidonic acids formed by oxygenation of 5S-HETE by native and aspirin-acetylated COX-2

Biosynthesis of the prostaglandin endoperoxide by the cyclooxygenase (COX) enzymes is accompanied by formation of a small amount of 11R-hydroxyeicosatetraenoic acid (HETE), 15R-HETE, and 15S-HETE as by-products. Acetylation of COX-2 by aspirin abrogates prostaglandin synthesis and triggers formation of 15R-HETE as the sole product of oxygenation of arachidonic acid. Here, we investigated the formation of by-products of the transformation of 5S-HETE by native COX-2 and by aspirin-acetylated COX-2 using HPLC-ultraviolet, GC-MS, and LC-MS analysis. 5S,15S- dihydroxy (di)HETE, 5S,15R-diHETE, and 5S,11R-diHETE were identified as by-products of native COX-2, in addition to the previously described di-endoperoxide (5S,15S-dihydroxy-9S,11R,8S,12S-diperoxy-6E,13E-eicosadienoic acid) as the major oxygenation product. 5S,15R-diHETE was the only product formed by aspirin-acetylated COX-2. Both 5,15-diHETE and 5,11-diHETE were detected in CT26 mouse colon carcinoma cells as well as in lipopolysaccharide-activated RAW264.7 cells incubated with 5S-HETE, and their formation was attenuated in the presence of the COX-2 specific inhibitor, NS-398. Aspirin-treated CT26 cells gave 5,15-diHETE as the most prominent product formed from 5S-HETE. 5S,15S-diHETE has been described as a product of the cross-over of 5-lipoxygenase (5-LOX) and 15-LOX activities in elicited rat mononuclear cells and human leukocytes, and our studies implicate cross-over of the 5-LOX and COX-2 pathways as an additional biosynthetic route.

A Mouse Mutation in the 12R-Lipoxygenase, Alox12b, Disrupts Formation of the Epidermal Permeability Barrier

Nonbullous congenital ichthyosiform erythroderma (NCIE) is a nonsyndromic form of autosomal recessive congenital ichthyosis characterized by hyperkeratosis and a disruption in the epidermal permeability barrier. Identification of mutations in two lipoxygenases (LOXs), ALOX12B (12R-LOX) and ALOXE3 (eLOX3), and further functional studies implicate ALOX12B and ALOXE3 in the etiology of NCIE. Here, we report a mutation in Alox12b in the recessive ethylnitrosurea-induced mouse mutant, mummy (Alox12b(mmy-Bei)). mummy mutants have red, scaly skin and die perinatally. Histologically, mummy mutants display defects in the epidermis. We mapped mummy to a 1.9 Mb interval on Chr. 11 containing Alox12b (12R-LOX), Aloxe3 (eLOX3) and Alox15b (8-LOX). Sequencing of all three genes identified a nonsense mutation in the catalytic domain of Alox12b. We demonstrate that mummy mutants have a disrupted epidermal permeability barrier and that the nonsense mutation in mummy abolishes the enzyme activity of 12R-LOX. The mummy mutant provides a mouse model for LOX-mediated NCIE and is the first described mouse mutant affecting epidermal barrier formation identified by forward genetics.

Chemical Determinants Involved in Anandamide-induced Inhibition of T-type Calcium Channels

Anandamide, originally described as an endocannabinoid, is the main representative molecule of a new class of signaling lipids including endocannabinoids and N-acyl-related molecules, eicosanoids, and fatty acids. Bioactive lipids regulate neuronal excitability by acting on G-protein-coupled receptors (such as CB1) but also directly modulate various ionic conductances including voltage-activated T-type calcium channels (T-channels). However, little is known about the properties and the specificity of this new class of molecules on their various targets. In this study, we have investigated the chemical determinants involved in anandamide-induced inhibition of the three cloned T-channels: Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3. We show that both the hydroxyl group and the alkyl chain of anandamide are key determinants of its effects on T-currents. As follows, T-currents are also inhibited by fatty acids. Inhibition of the three Ca(V)3 currents by anandamide and arachidonic acid does not involve enzymatic metabolism and occurs in cell-free inside-out patches. Inhibition of T-currents by fatty acids and N-acyl ethanolamides depends on the degree of unsaturation but not on the alkyl chain length and consequently is not restricted to eicosanoids. Inhibition increases for polyunsaturated fatty acids comprising 18-22 carbons when cis-double bonds are close to the carboxyl group. Therefore the major natural (food-supplied) and mammalian endogenous fatty acids including gamma-linolenic acid, mead acid, and arachidonic acid as well as the fully polyunsaturated omega3-fatty acids that are enriched in fish oil eicosapentaenoic and docosahexaenoic acids are potent inhibitors of T-currents, which possibly contribute to their physiological functions.

Acyl‐based anandamide uptake inhibitors cause rapid toxicity to C6 glioma cells at pharmacologically relevant concentrations

Compounds blocking the uptake of the endogenous cannabinoid anandamide (AEA) have been used to explore the functions of the endogenous cannabinoid system in the CNS both in vivo and in vitro. In this study, the effects of four commonly used acyl-based uptake inhibitors [N-(4-hydroxyphenyl)arachidonylamide (AM404), N-(4-hydroxy-2-methylphenyl) arachidonoyl amide (VDM11), (5Z,8Z,11Z,14Z)-N-(3-furanylmethyl)-5,8,11,14-eicosatetraenamide (UCM707) and (9Z)-N-[1-((R)-4-hydroxybenzyl)-2-hydroxyethyl]-9-octadecen-amide (OMDM2)] and the related compound arvanil on C6 glioma cell viability were investigated. All five compounds reduced the ability of the cells to accumulate calcein, reduced the total nucleic acid content and increased the activity of lactate dehydrogenase recovered in the cell medium. AM404 (10 microm) and VDM11 (10 microm) acted rapidly, reducing cell viability after 3 h of exposure when cell densities of 5,000 per well were used. In contrast, UCM707 (30 microm), OMDM2 (10 microm) and the related compound arvanil (10 microm) produced a more slowly developing effect on cell viability, although robust effects were seen after 6-9 h of exposure. At higher cell densities, the toxicities of AM404 and UCM707 were reduced. Comparison of the compounds with arachidonic acid, arachidonic acid methyl ester, AEA, arachidonoyl glycine and oleic acid suggested that the toxicity of the arachidonoyl-based compounds was related primarily to the acyl side-chain rather than the head group. A variety of pre-treatments blocking possible metabolic pathways and receptor targets were tested, but the only consistent protective treatment against the effects of these compounds was the antioxidant N-acetyl-L-cysteine. It is concluded that AM404, VDM11, UCM707 and OMDM2 produce a rapid loss of C6 glioma cell viability over the same concentration range as is required for the inhibition of AEA uptake in vitro, albeit with a longer latency. Such effects should be kept in mind when acyl-derived compounds are used to probe the function of the endocannabinoid system in the CNS, particularly in chronic administration protocols.

Differential regulation by fatty acids of protein histidine phosphorylation in rat pancreatic islets

Long-chain fatty acids (e.g. arachidonic acid) have been implicated in physiological control of insulin secretion. We previously reported histidine phosphorylation of at least two islet proteins (e.g., NDP kinase and the beta subunit of trimeric G-proteins), and suggested that such a signalling step may have regulatory roles in beta cell signal transduction, specifically at the level of G-protein activation. Since our earlier findings also indicated potential regulation by long-chain fatty acids of islet G-proteins, we undertook the current study to verify putative regulation, by fatty acids, of protein histidine phosphorylation of NDP kinase and Gbeta subunit in normal rat islets. The phosphoenzyme formation of NDP kinase was stimulated by various fatty acids in the following rank order: linoleic acid > arachidonic acid > oleic acid > palmitic acid = stearic acid = control. Furthermore, the catalytic activity of NDP kinase was stimulated by these fatty acids in the rank order of: oleic acid > arachidonic acid > linoleic acid > palmitic acid = stearic acid = control. Arachidonic acid methyl ester, an inactive analog of arachidonic acid, did not significantly affect either the phosphoenzyme formation or the catalytic activity of NDP kinase. Interestingly, arachidonic acid exerted dual effects on the histidine phosphorylation of beta subunit; it significantly stimulated the phosphorylation at 33 microM beyond which it was inhibitory. Together, these findings identify additional loci (e.g., NDP kinase and Gbeta subunit) at which unsaturated, but not saturated, fatty acids could exert their intracellular effects leading to exocytotic secretion of insulin.

Inhibition of C6 glioma cell proliferation by anandamide, 1-arachidonoylglycerol, and by a water soluble phosphate ester of anandamide: variability in response and involvement of arachidonic acid

It has previously been shown that the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) inhibit the proliferation of C6 glioma cells in a manner that can be prevented by a combination of capsazepine (Caps) and cannabinoid (CB) receptor antagonists. It is not clear whether the effect of 2-AG is due to the compound itself, due to the rearrangement to form 1-arachidonoylglycerol (1-AG) or due to a metabolite. Here, it was found that the effects of 2-AG can be mimicked with 1-AG, both in terms of its potency and sensitivity to antagonism by Caps and CB receptor antagonists. In order to determine whether the effect of Caps could be ascribed to actions upon vanilloid receptors, the effect of a more selective vanilloid receptor antagonist, SB366791 was investigated. This compound inhibited capsaicin-induced Ca 2+ influx into rVR1-HEK293 cells with a p K B value of 6.8±0.3. The combination of SB366791 and CB receptor antagonists reduced the antiproliferative effect of 1-AG, confirming a vanilloid receptor component in its action. 1-AG, however, showed no direct effect on Ca 2+ influx into rVR1-HEK293 cells indicative of an indirect effect upon vanilloid receptors. Identification of the mechanism involved was hampered by a large inter-experimental variation in the sensitivity of the cells to the antiproliferative effects of 1-AG. A variation was also seen with anandamide, which was not a solubility issue, since its water soluble phosphate ester showed the same variability. In contrast, the sensitivity to methanandamide, which was not sensitive to antagonism by the combination of Caps and CB receptor antagonists, but has similar physicochemical properties to anandamide, did not vary between experiments. This variation greatly reduces the utility of these cells as a model system for the study of the antiproliferative effects of anandamide. Nevertheless, it was possible to conclude that the antiproliferative effects of anandamide were not solely mediated by either its hydrolysis to produce arachidonic acid or its CB receptor-mediated activation of phospholipase A 2 since palmitoyltrifluoromethyl ketone did not prevent the response to anandamide. The same result was seen with the fatty acid amide hydrolase inhibitor palmitoylethylamide. Increasing intracellular arachidonic acid by administration of arachidonic acid methyl ester did not affect cell proliferation, and the modest antiproliferative effect of umbelliferyl arachidonate was not prevented by a combination of Caps and CB receptor antagonists.

Calcium-independent phospholipase A(2)-derived arachidonic acid is essential for endothelium-dependent relaxation by acetylcholine.

The role of calcium-independent phospholipase A(2) (iPLA(2))-produced arachidonic acid (AA) in acetylcholine (ACh)-mediated, endothelium-dependent vascular relaxation was investigated. ACh-induced relaxation of phenylephrine-constricted isolated rat mesenteric resistance arteries was attenuated following pretreatment with (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL; 1 microM; p < 0.01), a highly selective suicide substrate inhibitor of iPLA(2). Following BEL, the ACh relaxation could be completely restored following pretreatment with picomolar quantities of the cell-permeant methyl ester analog of AA (arachidonic acid methyl ester, AA-Me). Higher amounts of AA-Me (1 microM) had a direct endothelium-dependent relaxing action, which was inhibited by the nitric-oxide synthase inhibitor (N(omega)-nitro-L-arginine; 100 microM), independent of ACh, and unaffected by BEL. Neither the ACh relaxation restoring action nor the direct relaxing action of AA-Me was affected by preincubation with inhibitors of the lipoxygenase (esculetin, 10 microM) or cytochrome P450 monooxygenase (17-octadecynoic acid; 10 microM) pathways; and both actions of AA-Me were enhanced following preincubation with the cyclooxygenase inhibitor indomethacin (10 microM; p < 0.05). The results of the present study indicate that iPLA(2)-produced AA plays an essential role in ACh-mediated endothelium-dependent relaxation in rat mesenteric resistance arteries.

Arachidonic acid-induced H+ and Ca2+ increases in both the cytoplasm and nucleoplasm of rat cerebellar granule cells.

1. Arachidonic acid (AA) exerts multiple physiological and pathophysiological effects in the brain. By continuously measuring the intracellular pH (pH(i)) and Ca2+ levels ([Ca2+]i) in primary cultured rat cerebellar granule cells, we have found, for the first time, that 20 min treatment with 10 microM AA resulted in marked increases in Ca2+ and H+ levels in both the cytosol and nucleus. 2. A much higher concentration (40 mM) of another weak acid, propionic acid, was needed to induce a similar change in pH(i). The [Ca2+]i increase was probably caused by AA-induced activation of Ni2+-sensitive cationic channels, but did not involve NMDA channels or the Na+-Ca2+ exchanger. 3. AA-induced acidosis occurs by a different mechanism involving predominantly the passive diffusion of the un-ionized form of AA, rather than a protein carrier, as proposed by Kamp & Hamilton for fatty acids (FAs) in artificial phospholipid bilayers (the 'flip-flop' model). The following results, which are similar to those observed in lipid bilayers, support this conclusion: (1) FAs containing a -COOH group (AA, linoleic acid, alpha-linolenic acid, and docosahexaenoic acid) induced intracellular acidosis, whereas a FA with a -COOCH3 group (AA methyl ester) had little effect on pH(i), (2) a FA amine, tetradecylamine, induced intracellular alkalosis, and (3) the AA-/FA-induced pH(i) changes were reversed by bovine serum albumin. 4. Further evidence in support of a passive diffusion model, rather than a membrane protein carrier, is that: (1) there was a linear relationship between the initial rate of acid flux and the concentration of AA (2-100 microM), (2) acidosis was not inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, a potent inhibitor of the plasma membrane FA carrier protein, and (3) the involvement of most known H+-related membrane carriers and H+ conductance has been ruled out. 5. Since AA can be released under both physiological and pathophysiological conditions, the possible significance of the AA-evoked increases in H+ and Ca2+ in both the cytoplasm and nucleoplasm is discussed.

Transformation of phospholipid membranes by thiyl radicals via cis– trans isomerization of fatty acid residues

Thiyl radical-mediated transformation of unsaturated fatty acid residues are reported. Beside the initiation of lipid peroxidation, thiyl radicals can efficiently cause isomerization of fatty acid residues in a catalytic manner. The latter process is observed in homogeneous solutions as well as in organized assemblies, leading to a denaturation of the natural all- cis-isomers of fatty acid residues of lipid bilayers. The degree of denaturation goes parallel with an increase in the number of double bonds and in the hydrophobicity of thiols. For arachidonic acid methyl ester, a decay of the all- cis-isomer to approximately 30% after a gamma irradiation dose of 1.8 kGy in micelles is observed. The reversibility of the isomerization reaction is demonstrated in liposomes by the fact that the cis/ trans-equilibrium is independent of configuration of the starting compound.

Possible mechanism(s) of arachidonic acid-induced intracellular acidosis in rat cardiac myocytes.

Arachidonic acid (AA) and other nonesterified fatty acids (FAs) have been shown to exert harmful effects during cardiac ischemia. By continuously measuring intracellular pH (pH(i)) changes in neonatal and adult cardiac myocytes, we have found, for the first time, that 10 micromol/L AA induces a substantial intracellular acidosis (0.3 to 0.4 pH units). We have ruled out the possibilities that the AA-induced acidosis is caused by (1) inhibition or stimulation of the pH(i) regulators, (2) protein kinase C activation or the generation of AA metabolites or free radicals, or (3) activation of NADPH oxidase or an inward H(+) current. The AA-induced acidosis fits to a simple diffusion mechanism, as proposed by Kamp and Hamilton (flip-flop model) for artificial phospholipid bilayers. The important properties found in the cardiac myocyte are that (1) the initial rate of acid flux (J(H)) increases with the AA concentration (2 to 50 micromol/L), (2) FAs with a (-)COOH group (eg, AA, oleic acid, and linoleic acid) induce intracellular acidification, but FAs with a (-)COOCH(3) group (eg, AA methyl ester) have little effect on the pH(i), (3) tetradecylamine (FA amine) induces intracellular alkalosis, and, most importantly, (4) both the AA- and tetradecylamine-induced pH(i) changes can be reversed by 0.3% BSA. Because a low concentration of AA (10 micromol/L) can induce a substantial acidosis, the possible involvement of the FA-evoked acidosis in the negative inotropic effect during cardiac ischemia is discussed. The full text of this article is available at http://www. circresaha.org.

Ca2+/Calmodulin-independent Activation of Calcineurin from Dictyostelium by Unsaturated Long Chain Fatty Acids

This study describes a novel mode of activation for the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin. Using purified calcineurin from Dictyostelium discoideum we found a reversible, Ca(2+)/calmodulin-independent activation by the long chain unsaturated fatty acids arachidonic acid, linoleic acid, and oleic acid, which was of the same magnitude as activation by Ca(2+)/calmodulin. Half-maximal stimulation of calcineurin occurred at fatty acid concentrations of approximately 10 microM with either p-nitrophenyl phosphate or RII phosphopeptide as substrates. The methyl ester of arachidonic acid and the saturated fatty acids palmitic acid and arachidic acid did not activate calcineurin. The activation was shown to be independent of the regulatory subunit, calcineurin B. Activation by Ca(2+)/calmodulin and fatty acids was not additive. In binding assays with immobilized calmodulin, arachidonic acid inhibited binding of calcineurin to calmodulin. Therefore fatty acids appear to mimic Ca(2+)/calmodulin action by binding to the calmodulin-binding site.