Arachidonic acid release from diacyl phosphatidylethanolamine by human platelet membranes.

At pH9.5 in the presence of 10 mM-Ca2+, human platelet membranes released 22% (167 of 785 nmol) of arachidonic acid that was esterified to phospholipids. With the use of synthetic choline (dinonadecanoyl) and ethanolamine (diheptadecanoyl) phosphoglycerides as internal reference compounds, 115 nmol of the released arachidonic acid was shown to be derived from endogenous breakdown of the phosphatidylethanolamine fraction. Further, the lysophosphatidylethanolamine that was released along with the arachidonic acid was shown virtually to lack fatty aldehydes and to contain a preponderance of fatty acids that have a preference for esterification at the 1-position of naturally occurring phosphatidylethanolamine of human platelets. These findings ruled out plasmalogen phosphatidylethanolamine as the source of the released arachidonic acid. We conclude that diacyl phosphatidylethanolamine was the principal source of arachidonic acid released by human platelet membranes under the conditions described.     

Mechanism of modulation of the plasma membrane Ca(2+)-ATPase by arachidonic acid

The intracellular level of long chain fatty acids controls the Ca(2+) concentration in the cytoplasm. The molecular mechanisms underlying this Ca(2+) mobilization are not fully understood. We show here that the addition of low micromolar concentrations of fatty acids directly to the purified plasma membrane Ca(2+)-ATPase enhance ATP hydrolysis, while higher concentration decrease activity, exerting a dual effect on the enzyme. The effect of arachidonic acid is similar in the presence or absence of calmodulin, acidic phospholipids or ATP at the regulatory site, thereby precluding these sites as probable acid binding sites. At low arachidonic acid concentrations, neither the affinity for calcium nor the phosphoenzyme levels are significantly modified, while at higher concentrations both are decreased. The action of arachidonic acid is isoenzyme specific. The increase on ATP hydrolysis, however, is uncoupled from calcium transport, because arachidonic acid increases the permeability of erythrocyte membranes to calcium. Oleic acid has no effect on membrane permeability while linoleic acid shows an effect similar to that of arachidonic acid. Such effects might contribute to the entry of extracellular Ca(2+) following to fatty acid release.     

Effect of sex and gonadal hormones on rat plasma lipids during the development of an essential fatty acid deficiency

1. Male, female and castrated rats treated with oestradiol (30mug./week) or testosterone (2mg./week) were given an essential fatty acid-deficient diet containing 10% of hydrogenated coconut oil for 9 weeks. The concentrations and fatty acid composition of plasma phospholipids, cholesteryl esters and triglycerides were determined. 2. Between the second and third weeks of the deficiency, concentrations of plasma cholesteryl esters, phospholipids and triglycerides decreased, then remained relatively constant. There were no significant differences between males and females, but oestradiol caused a significant rise in plasma phospholipids and triglycerides as compared with testosterone-treated animals. 3. During the first 2 weeks of the deficiency, linoleic acid in the plasma lipids of all groups decreased to low concentrations and changed very little thereafter. 4. Female rats maintained higher percentages and concentrations of arachidonic acid and stearic acid in plasma phospholipids and arachidonic acid in cholesteryl esters than did males. Males had higher proportions of eicosatrienoic acid and oleic acid. There was no sex difference in the fatty acid composition of plasma triglycerides. 5. Oestradiol-treated rats had concentrations of cholesteryl and phospholipid arachidonate comparable with those of female rats and higher than the testosterone-treated group. Eicosatrienoic acid in the oestradiol-treated rats was high and resembled that of the male rats, apparently because of the higher concentration of plasma phospho lipids in this group. 6. Supplementation of the essential fatty acid-deficient rats with linoleate restored plasma cholesteryl and phospholipid linoleate and arachidonate nearly to normal concentrations in a single day. The increase in arachidonic acid in these fractions was accompanied by a similar quantitative decrease in eicosatrienoic acid. 7. These sex differences appear to be related to the smaller size of the female rat and to a more direct influence of oestradiol on the formation or maintenance of phospholipids rich in arachidonic acid.     

Determination of trans-arachidonic acid isomers in human blood plasma

Endogenous trans fatty acids originate from diet, but recent studies also suggest that cis-trans isomerization of fatty acids is possible by nitrogen dioxide radical, a product of NO and nitrite oxidation. We developed a method for quantitative analysis of four trans-arachidonic acids (TAA) in human plasma using isotopic dilution gas chromatography/mass spectrometry (GC/MS) with deuterium-labeled internal standard. Esterification of the plasma fatty acid extract with pentafluorobenzyl (PFB) bromide followed by high-performance liquid chromatography purification yielded a fairly pure fraction containing TAA-PFB esters that was analyzed by GC/MS. Partial separation of the TAA isomers was obtained on various GC columns. Comparison of the retention time with the synthetic standards revealed that all four TAA isomers are present in human plasma. The mean concentration of TAA in human plasma was 20.2ng/ml. The levels of isomers were 12.48+/-1.28, 2.75+/-0.39, and 4.99+/-0.74ng/ml for 5E-AA + 11E-AA, 8E-AA, and 14E-AA, respectively. The identification of TAA in plasma suggests that isomerization of arachidonic acid occurs in vivo. Our method allows distinguishing between the dietary and the NO(2)-dependent mechanisms of trans fatty acid formation and will be useful in defining the role of TAA as an in vivo marker of nitrooxidative stress in clinical and experimental settings.     

Turnover of plasma oleic acid measured by radio-gas chromatography.

Gas-liquid chromatography with radioactivity detection (Radio-GLC) was investigated as an analytical means of determining the fractional turnover rates of plasma free fatty acids. For this purpose normal dogs were infused with 1.838 muCi/min of [1-14C]oleic acid complexed with albumin and plasma samples were taken at 0 to 110 minutes. The plasma free fatty acids were isolated by a modified Dole extraction and the methyl esters, prepared by diazomethylation, were identified and quantitated by GLC and radio-GLC using radioactive methyl heptadecanoate as internal standard. The study demonstrates that physiologically feasible infusion rates and loads of radioactive acids can be found which permit accurate analyses of plasma free fatty acids by radio-GLC. During a 2-hour infusion no labeled acid other than oleic appeared in plasma indicating that the method could be used to study the turnover of a mixture of fatty acids simultaneously. These results also indicate that conventional methods of determination of radioactivity in purified extracts can be employed without concern for recycling of label among the fatty acids, at least over short periods of time. The radio-GLC technique described yields approximately 20% higher fractional turnover times for oleic acid than do standard methods.     

Patterns of fatty acid release from endogenous substrates by human platelet homogenates and membranes.

We describe a method for measuring the release of fatty acids from endogenous substrates of human platelet homogenates and membranes. The method depends on the availability of lipids whose fatty acids are odd-chained and therefore suitable as internal reference compounds that, at the time of lipid extraction, can be added to an incubation to permit subsequent quantification of the content of free fatty acids or fatty acids esterified to specific lipids. We found four types of lipolytic activities in human platelets. In homogenates at pH 4.0 a triglyceride lipase operated as shown by the synchrony of triglyceride degradation and release of glycerol and those fatty acids that are the predominant constituents of triglycerides. However, enough arachidonic acid was released at this pH level to suggest some phospholipid breakdown, since triglycerides hold relatively small amounts of this acid. With membranous preparations, in the alkaline pH range there were two peaks of fatty acid release with accompanying degradation of phospholipids. At pH 8.5, where release of the saturated acids, palmitic and stearic, predominated, their sum was 3.5 times that of arachidonic acid. At pH 9.5 the release of palmitic and stearic acids was only slightly below their peak values; however, the release of arachidonic acid nearly equaled the sum of the saturated acids. Linoleic acid was not released in representative amounts by those reactions that released arachidonic acid, despite the overwhelming propensity of both to be esterified at the 2-position of phospholipids. Pertinently, the choline phospholipids are linoleic-rich and the non-choline phospholipids linoleic-poor, while both have a generous endowment of arachidonic acid. With this in mind, we raise the possibility that the phospholipase A2 of human platelets is an endoenzyme because of its tendency to act on those phospholipids that are thought to comprise the inner layer of the cell membrane.     

Effects of postdecapitation ischemia on the metabolism of [14C]arachidonic acid and [14C]palmitic acid in the mouse brain

The effect of postdecapitation ischemia on the labeling of the free fatty acid pool and their incorporation in lipids was examined during the first 10 min after decapitation in mouse brain that had been injected intracerebrally with either [1-14C]arachidonic acid or [1-14C]palmitic acid. One min after decapitation, animals injected with labeled arachidonic acid exhibited a greatly reduced incorporation of label in brain phospholipids, diglycerides, and triglycerides. When radioactive palmitic acid was used, brain lipids exhibited considerably less inhibition of label. However, a similar degree of inhibition was observed 10 min after decapitation with both fatty acids. At this time, free arachidonic acid had decreased 84% as compared to the 24% decrease observed in the controls, and about 77% of the free palmitic acid remained in the free fatty acid fraction as compared with 30% in the controls. This decreased labeling may reflect ATP shortage that affects the fatty acid activation-reacylation reactions or the enzymes involved. Alternatively, the enhanced endogenous free arachidonic acid may compete with the radiolabeled arachidonic acid resulting in an inhibition of lipid labeling. Inhibition of label may have been greater in radiolabeled arachidonic acid than palmitic because of the larger accumulation of the former endogenous fatty acid during early ischemia.     

Elimination of n-butylated hydroxytoluene methylation during fatty acid analysis by gas chromatography

An improved method for fatty acids analysis with optimum recovery of highly polyunsaturated fatty acids methyl esters in biological systems is presented. The method is based on transesterification of phospholipid and triacylglycerols to fatty acid methyl esters using a commercially available reagent, Methyl-Prep II. Without proper precautions, as much as 50% of n-butylated hydroxytoluene (BHT) added to prevent oxidation of polyunsaturated fatty acids, could be methylated during the transesterification step. Methylated BHT elutes close to 14:0 (myristic acid) and no longer functions as an antioxidant, but the modified conditions virtually eliminate the methylation of BHT. Sample extraction and methylation was completed in 30 min at room temperature. A chelator (diethylenetriamine-pentaacetic acid; DTPA) is also added to prevent peroxidation of metal catalyzed free radical chain reactions. The standard deviations of the major fatty acids from multiple human plasma samples prepared on different days were less than 5%. The recovery of arachidonic acid, 20:4, from plasma was improved using the new method, and the recovery for docosahexaenoic acid, 22:6, spiked to human plasma was found to be 99%.     

Quantitation of the fatty acid composition of phosphatidic acid by capillary gas chromatography electron-capture detection with picomole sensitivity

We describe a relatively simple and sensitive method to measure fentomole amounts of phosphatidic acid in cells. Phosphatidic acid was extracted from cells in the presence of 1-heptadecanoyl-2-heptadecanoyl-sn-glycero-3-phosphate as an internal standard, purified by two-dimensional thin-layer chromatography, and hydrolyzed to its constituent free fatty acids which were then derivatized to the corresponding pentafluorobenzyl esters. Pentafluorobenzyl esters of fatty acids were analyzed by gas chromatography with electron-capture detection. Long-chain fatty acids were resolved with excellent signal-to-noise ratios. Using heptadecanoic acid as an internal standard for quantitation, as little as 1 fmol of pentafluorobenzyl ester of stearic acid was detected with a linear response up to 10 pmol. Linear detector responses were obtained for all major classes of fatty acids. For phosphatidic acid measurement, the detection limit was at least 50 fmol thus achieving a 1000-fold increase in sensitivity compared to the most sensitive of the previously described methods. An example is provided of quantitating phosphatidic acid from minute amounts of biological samples such as islets of Langerhans.     

Fusion of lamellar body with plasma membrane is driven by the dual action of annexin II tetramer and arachidonic acid

Annexin II has been implicated in membrane fusion during the exocytosis of lamellar bodies from alveolar epithelial type II cells. Most previous studies were based on the fusion assays by using model membranes. In the present study, we investigated annexin II-mediated membrane fusion by using isolated lamellar bodies and plasma membrane as determined by the relief of octadecyl rhodamine B (R18) self-quenching. Immunodepletion of annexin II from type II cell cytosol reduced its fusion activity. Purified annexin II tetramer (AIIt) induced the fusion of lamellar bodies with the plasma membrane in a dose-dependent manner. This fusion is Ca2+-dependent and is highly specific to AIIt because other annexins (I and II monomer, III, IV, V, and VI) were unable to induce the fusion. Modification of the different functional residues of AIIt by N-ethylmaleimide, nitric oxide, or peroxynitrite abolished AIIt-mediated fusion. Arachidonic acid enhanced AIIt-mediated fusion and reduced its Ca2+ requirement to an intracellularly achievable level. This effect is due to membrane-bound arachidonic acid, not free arachidonic acid. Other fatty acids including linolenic acid, palmitoleic acid, myristoleic acid, stearic acid, palmitic acid, and myristic acid had little effect. AIIt-mediated fusion was suppressed by the removal of arachidonic acid from lamellar body and plasma membrane using bovine serum albumin. The addition of arachidonic acid back to the arachidonic acid-depleted membranes restored its fusion activity. Our results suggest that the fusion between lamellar bodies with the plasma membrane is driven by the synergistic action of AIIt and arachidonic acid.     

The effect of essential fatty acid deficiency upon fatty acid uptake by the brain.

Young adult rats, either control or essential fatty acid deficient, were administered either [3-H] oleic acid or [3-H] arachidonic acid by stomach tube. In addition, a group of control rats was given [3-H] palmitic acid. The rats were killed at various times therafter, and the radioactivity of the lipids of brain and plasma was examined. In confirmation of previous work, the blood lipid label was found to rise rapidly and then fall, wheras the activity of brain lipids increased slowly and did not show a decline through the 24-h period studied. Analysis of the brain uptake data according to first-order kinetics confirmed the impressions gained from visual inspection of the data. The initial rate of uptake of arachidonic acid was about 4.5 times that of oleic acid in control animals and in deficient animals. Essential fatty acid deficiency, however, did not induce an altered rate of uptake for either oleic acid or arachidonic acid. The rate of uptake of palmitic acid by control rats was not significantly different from that of oleic acid. Even though the initial rates of incorporation of oleic and arachidonic acids were not changed during essential fatty acid deficiency, the final levels of radioactivity obtained in brain lipids were higher in deficient rats with both fatty acids. The plateau value obtained with oleic acid was 1.5 times higher in deficient animals, while the plateau value for arachidonic acid was 1.7 times higher. An experiment in which deficient animals were allowed access to a control diet for 12 or 24 h prior to the labeling experiment suggested that the higher levels of radioactivity found in brain lipids of deficient animals was not due to an isotope dilution effect. Such animals still displayed the labeling pattern of deficient animals with arachidonic acid, while the results with oleic acid varied somewhat. Our results suggest that essential fatty acid deficiency does not alter the ability of the brain to take up the fatty acids studied. However, the fatty acids, especially arachidonic, are retained in the brain to a greater extent in the deficient animals.     

Fatty acid changes in liver and plasma lipid fractions after safflower oil was fed to rats deficient in essential fatty acids

1. Fatty acid patterns of liver and plasma triglycerides, phospholipids and cholesteryl esters were determined at intervals during 24hr. after essential fatty acid-deficient rats were given one feeding of linoleate (as safflower oil). 2. Liver triglyceride, phospholipid and cholesteryl ester fatty acid compositions did not change up to 7hr. after feeding. Between 7 and 10hr., linoleic acid began to increase in all fractions, but arachidonic acid did not begin to rise in the phospholipid until 14-19hr. after feeding. 3. Oleic acid and eicosatrienoic acid in liver phospholipid began to decline at about the time that linoleic acid increased, i.e. about 9hr. before arachidonic acid began to increase. 4. Changes in linoleic acid, arachidonic acid and eicosatrienoic acid in phosphatidylcholine resembled those of the total phospholipid. Phosphatidylethanolamine had a higher percentage content of arachidonic acid before the linoleate was given than did phosphatidylcholine, and after the linoleate was given the fatty acid composition of this fraction was little changed. 5. The behaviour of the plasma lipid fatty acids was similar to that of the liver lipids, with changes in linoleic acid, eicosatrienoic acid and arachidonic acid appearing at the same times as they occurred in the liver. 6. The results indicated that linoleic acid was preferentially incorporated into the liver phospholipid at the expense of eicosatrienoic acid and oleic acid. The decline in these fatty acids apparently resulted from their competition with linoleic acid for available sites in the phospholipids rather than from any direct replacement by arachidonic acid.     

Arachidonic acid inhibits glycine transport in cultured glial cells.

The effects of arachidonic acid on glycine uptake, exchange and efflux in C6 glioma cells were investigated. Arachidonic acid produced a dose-dependent inhibition of high-affinity glycine uptake. This effect was not due to a simple detergent-like action on membranes, as the inhibition of glycine transport was most pronounced with cis-unsaturated long-chain fatty acids, whereas saturated and trans-unsaturated fatty acids had relatively little or no effect. Endogenous unsaturated non-esterified fatty acids may exert a similar inhibitory effect on the transport of glycine. The mechanism for this inhibitory effect has been examined in a plasma membrane vesicle preparation derived from C6 cells, which avoids metabolic or compartmentation interferences. The results suggest that part of the selective inhibition of glycine transport by arachidonic acid could be due to the effects of the arachidonic acid on the lipid domain surrounding the carrier.     

Fatty acid alterations and n-3 fatty acid supplementation in cystic fibrosis

Specific fatty acid alterations have been described in the blood and tissues of cystic fibrosis (CF) patients. The two most consistent alterations include decreased levels of linoleic acid (LA) and decreased levels of docosahexaenoic acid (DHA). Increased arachidonic acid (AA) release from membrane phospholipids, as well as changes in levels of AA and other monounsaturated and polyunsaturated fatty acids (PUFAs) have also been described in CF. Although mechanisms of fatty acid alterations have not yet been determined, these alterations may have an important role in the progression of the CF disease. There have been several clinical trials in which CF patients were supplemented with n-3 fatty acids. Most trials resulted in an increase in the levels of the supplemental fatty acids in the blood of CF patients in the absence of significant clinical improvement. It is recommended that future trials include a larger population of CF patients and measure multiple clinical outcomes.     

Fatty acid alterations and n-3 fatty acid supplementation in cystic fibrosis

Specific fatty acid alterations have been described in the blood and tissues of cystic fibrosis (CF) patients. The two most consistent alterations include decreased levels of linoleic acid (LA) and decreased levels of docosahexaenoic acid (DHA). Increased arachidonic acid (AA) release from membrane phospholipids, as well as changes in levels of AA and other monounsaturated and polyunsaturated fatty acids (PUFAs) have also been described in CF. Although mechanisms of fatty acid alterations have not yet been determined, these alterations may have an important role in the progression of the CF disease. There have been several clinical trials in which CF patients were supplemented with n-3 fatty acids. Most trials resulted in an increase in the levels of the supplemental fatty acids in the blood of CF patients in the absence of significant clinical improvement. It is recommended that future trials include a larger population of CF patients and measure multiple clinical outcomes.     

Arachidonic acid and linoleic acid supplementation increase prostanoid production in rats fed a butter-enriched diet

Male Sprague Dawley rats were fed a butter-enriched diet (50% fat) for 2 weeks and then supplemented orally with either 90 mg of ethyl arachidonate or ethyl linoleate daily for 2 weeks. For comparative reasons, one group of animals was fed standard laboratory rat chow for 4 weeks. Aortic prostacyclin (PGI2) production, platelet aggregation and thromboxane A2 (TXA2) production and plasma and aortic phospholipid (PL) fatty acids were measured. When compared to butter-fed rats, aortic PGI2 production, collagen-induced platelet aggregation and TXA2 production were significantly increased in rats supplemented with ethyl arachidonate to levels similar to those seen in chow-fed rats. Ethyl linoleate supplementation also tended to increase aortic PGI2 production, collagen-induced platelet aggregation and TXA2, but not to the same extent. These changes were accompanied by increases in the level of arachidonic acid and linoleic acid in aortic and plasma PL and a decrease in the level of eicosapentaenoic acid (EPA) and docsahexaenoic acid (DHA). These data indicate that supplementation with small doses of preformed arachidonic acid was more effective than supplementation with its precursor, linoleic acid, in reversing the effects on prostanoid production and phospholipid fatty acid composition in rats fed diets enriched with butter.     

Arachidonic acid, ARC channels, and Orai proteins

A critical role for arachidonic acid in the regulation of calcium entry during agonist activation of calcium signals has become increasingly apparent in numerous studies over the past 10 years or so. In particular, low concentrations of this fatty acid, generated as a result of physiologically relevant activation of appropriate receptors, induces the activation of a unique, highly calcium-selective conductance now known as the ARC channel. Activation of this channel is specifically dependent on arachidonic acid acting at the intracellular surface of the membrane, and is entirely independent of any depletion of internal calcium stores. Importantly, a specific role of this channel in modulating the frequency of oscillatory calcium signals in various cell types has been described. Recent studies, subsequent to the discovery of STIM1 and the Orai proteins and their role in the store-operated CRAC channels, have revealed that these same proteins are also integral components of the ARC channels and their activation. However, unlike the CRAC channels, activation of the ARC channels depends on the pool of STIM1 that is constitutively resident in the plasma membrane, and the pore of these channels is comprised of both Orai1 and Orai3 subunits. The clear implication is that CRAC channels and ARC channels are closely related, but have evolved to play unique roles in the modulation of calcium signals—largely as a result of their entirely distinct modes of activation. Given this, although the precise details of how arachidonic acid acts to activate the channels remain unclear, it seems likely that the specific molecular features of these channels that distinguish them from the CRAC channels – namely Orai3 and/or plasma membrane STIM1 – will be involved.     

Arachidonic acid as a second messenger. Interactions with a GTP-binding protein of human neutrophils.

Arachidonic acid (20:4) and other cis-unsaturated fatty acids exert direct effects on a variety of cells, effects that do not depend on the metabolism of fatty acids via cyclooxygenase or lipoxygenase pathways. In these studies arachidonic acid and other cis-unsaturated fatty acids (but not trans-unsaturated or saturated fatty acids) increased the specific binding of the nonhydrolyzable analog of GTP, [35S]GTP gamma S, to purified neutrophil membrane preparations and elicited superoxide anion generation from intact neutrophils. There was a positive correlation (r = 0.70) between the capacity of fatty acids to increase nucleotide binding and to elicit the respiratory burst. Scatchard plot analysis of binding at equilibrium demonstrated an increase in the number of available GTP binding sites in the presence of 50 microM arachidonic acid. Nonsteroidal antiinflammatory agents interfered with the arachidonic acid effect on [35S]GTP gamma S binding. ADP-ribosylation of the pertussis toxin substrate Gi alpha within the plasmalemma-reduced specific [35S]GTP gamma S binding and blocked arachidonate-dependent enhancement of binding. Moreover, pertussis toxin treatment of intact neutrophils inhibited arachidonic acid-induced superoxide anion generation. The data indicate that arachidonic acid directly activates a GTP binding protein in the neutrophil plasma membrane and may thereby act as a second messenger in signal transduction.     

Metmyoglobin promotes arachidonic acid peroxidation at acid pH

The ability of metmyoglobin and other heme proteins to promote peroxidation of arachidonic acid under acidic conditions was investigated. Incubation of metmyoglobin with arachidonic acid resulted in a pH-dependent increase in lipid peroxidation as measured by the formation of thiobarbituric acid reactive products and oxygen consumption. Increased peroxidation was observed at pH levels below 6.0, reaching a plateau between pH 5.5 and 5.0. At comparable heme concentrations, metmyoglobin was more efficient than oxymyoglobin, methemoglobin, or ferricytochrome c in promoting arachidonic acid peroxidation. Metmyoglobin also promoted peroxidation of 1-palmityl-2-arachidonyl phosphatidylcholine and methylarachidonate but at significantly lower rates than arachidonic acid. Addition of fatty acid-free albumin inhibited arachidonic acid peroxidation in a molar ratio of 6 to 1 (arachidonic acid:albumin). Both ionic and non-ionic detergents inhibited metmyoglobin-dependent arachidonic acid peroxidation under acidic conditions. The anti-oxidants butylated hydroxytoluene and nordihydroguaiaretic acid and low molecular weight compounds with reduced sulfhydryl groups inhibited the reaction. However, mannitol, benzoic acid, and deferoxamine were without significant effect. Visible absorption spectra of metmyoglobin following reaction with arachidonic acid showed minimal changes consistent with a low level of degradation of the heme protein during the reaction. These observations support the hypothesis that metmyoglobin and other heme proteins can promote significant peroxidation of unsaturated fatty acids under conditions of mildly acidic pH such as may occur at sites of inflammation and during myocardial ischemia and reperfusion. This may be the result of enhanced aggregation of the fatty acid and/or interaction of the fatty acid with heme under acidic conditions.     

Arachidonic acid potentiates exocytosis and allows neuronal SNARE complex to interact with Munc18a

Neuronal communication relies on the fusion of neurotransmitter-containing vesicles with the neuronal plasma membrane. Recent genetic studies have highlighted the critical role played by polyunsaturated fatty acids in neurotransmission, however, there is little information available about which fatty acids act on exocytosis and, more importantly, by what mechanism. We have used permeabilized chromaffin cells to screen various fatty acids of the n-3 and n-6 series for their acute effects on exocytosis. We have demonstrated that an n-6 series polyunsaturated fatty acid, arachidonic acid, potentiates secretion from intact neurosecretory cells regardless of the secretagogue used. We have shown that arachidonic acid dose dependently increases soluble NSF attachment protein receptor complex formation in chromaffin cells and bovine cortical brain extracts and that a non-hydrolysable analogue of arachidonic acid causes a similar increase in SNARE complex formation. This prompted us to examine the effect of arachidonic acid on SNARE protein interactions with Munc18a, a protein known to prevent Syntaxin1a engagement into the SNARE complex in vitro. In the presence of arachidonic acid, we show that Munc18a can interact with the neuronal SNARE complex in a dose-dependent manner. We further demonstrate that arachidonic acid directly interacts with Syntaxin1a.