Fatty Arachidonic Acid Research Articles

Thioesterification of platelet proteins with saturated and polyunsaturated fatty acids.

We have demonstrated that more than 20 platelet proteins can be acylated with fatty acids via thioester linkages. These include the glycoprotein IX beta chain of glycoprotein Ib, components of the von Willebrand factor receptor on the platelet surface, P-selectin, and alpha subunits of Gz, Gq, and Gi. Our studies have shown that platelet proteins can be posttranslationally acylated in thioester linkages not only with palmitate but with myristate and also with the eicosanoid precursor fatty acids arachidonate and eicosapentaenoate. Thioesterification of platelet proteins with fatty acids other than palmitate may have significant functional consequences for reversible binding of proteins to membranes.

Long-term fat intake and biomarkers

The patterns of fatty acids maintained in human tissues reflect the dietary abundances of n-3 and n-6 fatty acids and the selectivities of the various lipid-metabolizing enzymes and transport systems in tissues. Similar selectivities for enzymes of mice, rats, and humans permit data from all three to be used in predicting average long-term tissue responses to different diets. The participation of the highly unsaturated fatty acids arachidonate (an n-6 acid) and eicosapentaenoate (an n-3 acid) in chronic pathophysiological events gives importance to predicting their tissue abundance. The average proportions in tissue phospholipids are hyperbolically related to dietary supplies of their precursors linoleate (18:2n-6) and linolenate (18:3n-3). In contrast, the precursors are maintained in tissue triacylglycerols in a linear relation to their average dietary supply.

Copper and gas-phase cigarette smoke inhibit plasma lecithin:cholesterol acyltransferase activity by different mechanisms.

Cigarette smokers have reduced levels of plasma high density lipoprotein (HDL) compared to nonsmokers and are at risk of premature cardiovascular disease. Previous work from this laboratory has shown that exposure of human plasma to gas-phase cigarette smoke (CS) inhibited the activity of lecithin:cholesterol acyltransferase (LCAT), the enzyme that catalyzes the formation of cholesteryl ester in HDL and thereby promotes HDL maturation. As CS contains free radicals that could potentially oxidize plasma lipoproteins, we examined the involvement of lipid peroxidation in LCAT inhibition. Results obtained with CS were compared with those obtained by initiating lipid peroxidation with copper ions. Exposure of dialyzed human plasma to an equivalent of one-eighth of a cigarette at 15-min intervals resulted in a progressive loss of LCAT activity (50 and 90% reductions by 1 and 6 h, respectively). A similar pattern of LCAT inhibition was produced with copper (0.5 mM) where 50 and 97% reductions were observed at 1 and 6 h, respectively. To determine whether LCAT inhibition was related to lipid peroxidation, lipoprotein fractions corresponding to VLDL-IDL, LDL, and HDL were isolated from plasma exposed to CS or copper and analyzed for changes in TBARS, the polyunsaturated fatty acid arachidonate relative to palmitate (20:4/16:0 ratio), and vitamin E concentrations. Exposure of plasma for 6 h to CS had no effect on the levels of TBARS and 20:4/16:0 ratio; however, 6 h copper treatment (0.5 mM) caused a 3.0-, 4.0-, and 1.4-fold increase in TBARS and a 17, 25, and 13% reduction in the 20:4/16:0 ratio in VLDL-IDL, LDL, and HDL fractions, respectively. In addition, a complete depletion of lipoprotein vitamin E was observed with CS, whereas copper decreased vitamin E levels by approximately 50% in each fraction. Supplementation of plasma with either vitamin C (85 microM) or butylated hydroxytoluene (BHT, 0.45 mM) was unable to protect LCAT from CS. In contrast, BHT completely protected LCAT activity from inhibition by copper. We conclude that unlike copper, CS-induced inhibition of plasma LCAT activity was unrelated to free radical-induced lipid peroxidation. The inhibition of LCAT activity by cigarette smoke may contribute to the development of atherosclerosis by impairing HDL metabolism and the reverse cholesterol transport process.

Arachidonic fatty acid in red blood cell membranes, lymphocytes, and cultured skin fibroblasts of dominant Charcot-Marie-Tooth syndrome

Altered proportions of long-chain unsaturated n − 6 fatty acids (FA) in plasma and myelin of the heredodegenerative peripheral neuropathy, Charcot-Marie-Tooth syndrome (CMT), may implicate FA metabolism in the pathogenesis of CMT demyelination. A significant relationship between low plasma values of the polyunsaturated n − 6 FA, arachidonic acid, and an increased amount of prostaglandin-mediated immune responses had suggested the possibility of immune system metabolic usage of these n − 6 FA rather than an inherited FA enzyme defect. This relationship between peripheral immunostimulation and altered FA was documented repeatedly in CMT patients. Increased and cyclic lymphocyte activation expressions were measured at the same time as low amounts of plasma arachidonic acid, which is the FA precursor of prostaglandin immunoregulatory agents. An autoimmune process with possible enhanced formation of the n − 6 immune prostenoid agents in CMT had also been suggested by increased class II antigen expression on CMT sural nerves. Here, normal proportions of total FA in cultured CMT skin fibroblasts diminishes a notion of hereditary defects in CMT fatty acid metabolism. In addition, a significant depletion of the key arachidonic acid in lipid stores of CMT red blood cell membranes with elevated values of this FA in functional CMT lymphocytes, compared to controls, support the concept in CMT patients of a metabolic diversion of n − 6 FA, particularly arachidonic acid, from tissue stores for immunoregulatory prostenoid agent formation.

Potassium channels: Structure-function relationships, diversity, and pharmacology

There are many different types of potassium (K+) channels: A good number are voltage-dependent, others are activated by variations of intracellular concentrations of Ca2+ and the activity of others is controlled by cytoplasmic variations of the ATP/ADP ratio or by variations of intracellular Na+ or arachidonic acid and other fatty acids; a large number are modulated by phosphorylation and/or interaction with G proteins. Considerable progress has been made in the past few years in the molecular knowledge of some of these channels. Some of the voltage-dependent K+ channels have been cloned. In each tissue several genes encode several different K+ channel subunits that assemble to form large families of voltage-dependent K+ channels with different biophysical properties (different voltage dependence, different time course), which are associated with different physiological functions. The molecular structure of other types of K+ channels is not yet solved. Investigation of the molecular pharmacology of K+ channels has also made tremendous progress recently. High-affinity ligands are now available for some of the voltage-dependent K+ channels, Ca2+-activated K+ channels, and ATP-sensitive K+ channels.

Ischemia and reperfusion induced multilamellar vesicles in isolated rabbit hearts: Time correlation between morphometric data and metabolic alterations

In normoxic hearts a limited number of multilamellar vesicles was found in both endothelial cells and myocytes. The total number of multilamellar vesicles observed in myocytes, particularly those extruded from mitochondria, significantly increased in hearts rendered ischemic for at least 60 mins. The number of multilamellar vesicles extruded from sarcolemma was increased in hearts reperfused after this period of ischemia. The number of multilamellar vesicles in or adjacent to lipid droplets was independent of the duration of ischemia. Multilamellar vesicles were similar in size and periodicity of the lamellae. It is proposed that the number of multilamellar vesicles can be used to quantitate ischemic membrane injury. The formation of multilamellar vesicles was significantly related in time to (a) the accumulation of arachidonic acid and total fatty acids; (b) a decrease in the tissue content of ATP and (c) the release of lactate dehydrogenase (LDH). No significant alterations in the total tissue content of triacylglycerols and phospholipids were detected. The amount of arachidonic acid accumulated in the hearts reflects the degradation of only a minor fraction of the phospholipid pool. Assuming a close relationship between phospholipid degradation, induction of multilamellar vesicles and loss of cellular integrity, the present findings might indicate that the loss of a small part of phospholipids might have serious pathophysiological consequences, as indicated by the morphological changes in cellular membranes and the release of cytoplasmic macromolecules.

The effect of endogenous essential and nonessential fatty acids on the uptake and subsequent agonist-induced release of arachidonate

We have demonstrated that the uptake and agonist-induced release of a pulse of arachidonate are influenced by the size and composition of preexisting endogenous fatty acid pools. EFD-1 cells, an essential fatty acid-deficient mouse fibrosarcoma cell line, were incubated with radiolabeled (14C or 3H] arachidonate, linoleate, eicosapentaenoate (EPA), palmitate, or oleate in concentrations of 0-33 microM for 24 h. After 24 h, the cells were pulsed with 0.67 microM radiolabeled (3H or 14C, opposite first label) arachidonate for 15 min and then stimulated with 10 microM bradykinin for 4 min. Because EFD-1 cells contain no endogenous essential fatty acids, we were able to create essential fatty acid-repleted cells for which the specific activity of the newly constructed endogenous essential fatty acid pool was known. Loading the endogenous pool with the essential fatty acids arachidonate, eicosapentaenoate, or linoleate (15-20 nmol of fatty acid incorporated/10(6) cells) decreased the uptake of a pulse of arachidonate from 200 to 100 pmol/10(6) cells but had no effect on palmitate uptake. The percent of arachidonate incorporated during the pulse which was released upon agonist stimulation increased 2-fold (4-8%) as the endogenous pool of essential fatty acids was increased from 0 to 15-20 nmol/10(6) cells. This 8% release was at least 3-fold greater than the percent release from the various endogenous essential fatty acid pools. In contrast, loading the endogenous pool with the nonessential fatty acids oleate or palmitate to more than 2-3 times their preexisting cellular level had no effect on the uptake of an arachidonate pulse. Like the essential fatty acids, increasing endogenous oleate increased (by 2-fold) the percent release of arachidonate incorporated during the pulse, whereas endogenous palmitate had no effect on subsequent agonist-induced release from this arachidonate pool. These studies show that preexisting pools of essential and nonessential fatty acids exert different effects on the uptake and subsequent releasability of a pulse of arachidonate.

Eicosanoids and heart disease

The discovery of prostaglandins over 5 decades ago heralded a new era in the study of mediators or factors involved in physiological and pathophysiological processes. Prostaglandins, however, do not represent the sole products derived from arachidonic acid or other fatty acid precursors; instead these fatty acids can undergo metabolism to numerous bioactive compounds derived from cyclooxygenase, lipoxygenase, as well as the recently discovered epoxygenase cytochrome P-450 dependent pathways. Collectively, these products are commonly referred to as eicosanoids and have been implicated in numerous biological phenomena. It has long been hypothesized that the prostaglandins play an important role in cardiovascular regulation. For instance, the concept of a balance between the vasoconstrictor, prothrombotic properties of some arachidonic acid derived products and the opposing actions of others led to the development of drug therapies against thromboembolic disorders. Aspirin, for example, a well-known cyclooxygenase inhibitor, has been shown to be efficacious against myocardial infarction and stroke of embolic origin. Moreover, cyclooxygenase- and lipoxygenase-derived products have been implicated in various types of cardiac dysfunction including coronary constriction, arrhythmogenesis, anaphylactic reactions, or ischemic and reperfusion injury.In view of the evolving complexity of arachidonic acid metabolism and increasing evidence that eicosanoids, either alone, or through interaction with other substances, represent important mediators in either the development of heart disease or the myocardial response to injury, we organized this symposium entitled Eicosanoids and Heart Disease concurrent with the 31st Annual Meeting of the Canadian Federation of Biological Societies. A one-half day symposium precludes the possibility of detailed coverage of the many areas in cardiovascular disease in which eicosanoid participation could be implicated and which ideally one would hope to cover. The organizers' aim was to address, in the form of research presentations or reviews, current areas of investigation dealing with selected topics in heart disease.The success of this symposium was made possible by the participation of several distinguished scientists. We would also like to thank the Pharmacological Society of Canada, the Canadian Heart Foundation, Upjohn Canada, Sterling Drug, and Ciba-Geigy U.S. A. for financial support. The organizers thank Dr. J. Burka for serving as Guest Editor and Mrs. L. Hendrickson of this Journal for coordinating the submission of the manuscripts.

Effect of Excess Dietary Iron on Lipid Composition of Calf Liver, Heart, and Skeletal Muscle

Effect of excess dietary iron on lipid composition of calf liver, skeletal muscle, and heart was assessed. High dietary iron (5000 versus 100ppm in milk replacer DM) had no effect on the relative proportion of lipid classes in liver or their unsaturated fatty acid composition. In muscle some minor lipid components were reduced and cholesterol and sphingomyelin increased. Excessive iron had a marked effect, however, on heart lipid composition, reducing total lipids and almost all lipid classes; triglycerides, sphingomyelin, and lysophosphatidylcholine were increased. Characteristically, sphingomyelin increases in cell membranes in response to aging and numerous pathological conditions. High dietary iron reduced linolenic acid in phosphatidylethanolamine and phosphatidylcholine of both skeletal and cardiac muscle. This may have resulted from iron-caused ethane production from autoxidation of linolenic acid or other n-3 family fatty acids, an effect known to occur in the rat. Linoleic and arachidonic fatty acids appeared to be unaffected. Plasmalogens in muscle and heart phosphatidylethanolamine and phosphatidylcholine were increased by high iron intake. As these alk-1-enyl ethers protect cells from oxidation and radiation damage, their synthesis may have been increased in response to stress from excessive iron. The results indicate that a relatively high concentration of vitamin E may be required in calf milk replacer when excessive iron is present.

In vivo fluorescent probe study of zymosan-stimulated alveolar macrophages

UDC 612.112.95:576.314/.014.46.07 Under the influence of active forms of oxygen and other biooxidants, accumulating in the phagosomes of cells during a "respiratory burst," intensive oxidation of arachidonic and other polyunsaturated fatty acids of membrane phospholipids takes place via the cyclo-oxygen- ase pathway, with the formation of prostaglandins and their derivatives, and via the lipo- oxygenase pathway with synthesis of leukotrienes and their derivatives (6, 8). Meanwhile the topology and distribution of phospholipids in the membranes are drastically changed in ac- tivated M on account of acceleration of their resynthesis and breakdown (7). Transformation of the phospholipid matrix of biomembranes is manifested primarily as a change in their vis- cosity. Accordingly M can be regarded as a convenient model with which to study correlation between function of the cell and the state of its biomembranes.

Phospholipid metabolism of stimulated lymphocytes: Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [ 3H]arachidonate and [ 14C]oleate or [ 3H]arachidonate and [ 14C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3–7-fold) and phosphatidylethanolamine (2–3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.

Interactions between stimulated platelets and endothelial cells in vitro.

Prostaglandins and hydroxy acids are synthesized mainly from the essential polyunsaturated fatty acid arachidonate, and these substances have been identified in almost all mammalian tissues. Prostaglandins, thromboxane A2 (TXA2) and prostacyclin (PGI2) are autocoids that appear to function in the regulation of vascular tone, cell secretion and contractile processes. So far, hydroxy acids have been found to function as chemotactic agents and in the formation of slow-reacting substances. Other actions of hydroxy acids will certainly be defined in future research. The endoperoxides PGG2 and PGH2 represent common precursors of all prostaglandin end-products. In studying the prostaglandin metabolism of a specific tissue, the total profile of endoperoxide transformation should be determined. In platelets the endoperoxides are transformed mainly into TXA2, a potent vasoconstrictor and inducer of platelet aggregation. Endothelial cells convert endoperoxides to PGI2, a vasodilator and inhibitor of platelet aggregation. In addition, endothelial cells can utilize endoperoxides from stimulated plates to form PGI2. The concept that platelets and endothelial cells can share common precursors for the production of modulating substances may be applicable to other cell types.

Circulating Platelet Aggregates and Thrombocytopenia Induced by Intravenous Infusions of Arachidonic and Lauric Acids in Guinea Pigs

Slow intravenous infusion of Na laurate (NaL) into guinea pigs caused a rapid appearance of platelet aggregates in the arterial blood and a precipitous fall in platelet counts. During the infusion of Na arachidonate (NaA) thrombocytopenia developed slowly, and few and smaller platelet aggregates appeared in the arterial blood. Considerably more guinea pigs died during or after the NaA infusion than after administration of NaL. The possibility that arachidonic acid and other long-chain fatty acids may play a role in the development of thrombosis and thromboembolism is discussed.

Active Ca transport of sacroplasmic reticulum during experimental uremia. Changes in kinetics and lipid composition.

The Ca-transport system of sarcoplasmic vesicles of rabbits is altered by experimental uremia. 1. The influx rate constant of the experimental membranes decrease with a resulting decrease of the calcium influx rate. 2. The experimental membranes transport a smaller amount of Ca2+ per mol of ATP split than the controls, i.e. their transport ratio is discussed. 3. The calcium permeability of the experimental membranes increases with a resulting decreased concentrating ability. 4. The phosphatide content but not the cholesterol content of the experimental membranes decreases with a consequent increase of the cholesterol/phosphatide ratio. 5. The fatty acid pattern of total phosphatides of the experimental membranes changes. A relative decrease of palmitic acid and oleic acid occurs and a relative increase of stearic, arachidonic and higher unsaturated fatty acids. 6. The altered lipid composition of the membranes does not change the temperature dependence of the kinetics.

Effects of ethanol and a fat-free diet on hepatic mitochondrial fragility and fatty acid composition.

SummaryThe effect of EFA deficiency and chronic ethanol ingestion on liver mitochondrial fragility and fatty acid composition was studied using young growing male rats. The results indicated that EFA deficiency (fat-free diet) partially protected the mitochondria from developing the increased fragility caused by ethanol ingestion. The mitochondrial fragility induced by chronic ethanol feeding was not related to changes in the proportions of arachidonic acid or other mitochondrial fatty acids. Additionally, it was found that chronic ethanol feeding partially prevented the changes in the mitochondrial fatty acid composition induced by EFA deficiency.

Note on the Determinations of Blood Fat

The technique of Stewart and White1 for the determination of “blood fat” and its modifications by Himwich, Friedman and Spiers2 has yielded higher figures than have the methods developed by Stoddard and Drury,3 and Stewart, Gaddie and Dunlop.4 The essential technical difference of these methods is that in the Stewart and White1 procedure the liberated fatty acids are titrated directly, whereas, by the other methods the fatty acid precipitate is washed prior to titration. For this reason it is possible that the values obtained by the Stewart and White1 technique may include substances washed away in the other procedures. Certain fatty acids of the blood may be among those so washed away, for example, arachidonic and other unsaturated fatty acids. On the other hand, extraneous acids may also be lost in the process of washing, such as phosphoric or organic acids.The quantitative aspects of arachidonic and other unsaturated fatty acids of the blood are to be investigated in this laboratory. Stewart, Gaddie an...