Acids In Membrane Phospholipids Research Articles

Synthesis and action of eicosanoids in warm and cold-acclimated amphibians.

Eicosanoids, including prostaglandins (PGs) and leukotrienes (LTs) are derived from 20 carbon fatty acids in membrane phospholipids and are important physiological regulators in a variety of systems. While synthesis and actions of eicosanoids are well characterized in mammals, less is known about cellular production and biological actions in poikilothermic vertebrates. As body temperatures in poikilotherms reflect those of the environment, different acclimation states may therefore dramatically affect synthesis and action of eicosanoids. In cannulated bullfrogs, prostaglandins (Herman et al, 1986) and leukotrienes (Herman et al, 1991) have less potent cardiovascular effects in cold-acclimated (5°, 30 days) animals when compared with those at room temperature.KeywordsCold AcclimationAcid Ethyl EsterCarbon Fatty AcidEicosanoid SynthesisRana CatesbeianaThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Selective Inhibition of the Prothrombinase Complex: Factor Va Alters Macromolecular Recognition of a Tick Anticoagulant Peptide Mutant by Factor Xa,

The prothrombinase complex assembles through reversible interactions between the protease, factor Xa, the cofactor, factor Va, and acidic phospholipid membranes in the presence of calcium ions. Changes in macromolecular recognition by factor Xa which may result from its interaction with factor Va in the prothrombinase complex have been probed using a recombinant derivative of tick anticoagulant peptide where Arg3 has been replaced with Ala (R3A-TAP). In contrast to the wild type inhibitor, R3A-TAP was a weak competitive inhibitor of factor Xa (Ki = 794 nM). The inhibition of the prothrombinase complex by R3A-TAP was characterized by slow, tight-binding kinetics with an increased affinity of approximately 4000-fold (Ki* = 0.195 nM) relative to that of solution-phase factor Xa. Stopped-flow measurements using p-aminobenzamidine (PAB) demonstrated that the reaction between solution-phase factor Xa and R3A-TAP could be adequately described by a single reversible step with rate constants that were consistent with equilibrium binding measurements. The rate-limiting bimolecular combination of R3A-TAP and factor Xa was competitive with PAB binding of the protease. In contrast, the reaction of R3A-TAP with prothrombinase measured using PAB yielded biphasic stopped-flow traces, indicating a multistep pathway for the reaction of the inhibitor with the enzyme complex. The kinetic measurements were consistent with the initial formation of a ternary complex between R3A-TAP, prothrombinase, and PAB followed by two unimolecular steps which lead to PAB dissociation from the enzyme. In this case, prior occupation of the active site by PAB had no effect on the bimolecular reaction between R3A-TAP and prothrombinase. Thus, the interaction of factor Xa with factor Va on the membrane surface alters recognition of R3A-TAP by the protease, leading to changes in the thermodynamics as well as in the observed kinetic mechanism for the reaction. Therefore, a single amino acid substitution in TAP reveals large changes in macromolecular recognition by factor Xa as a consequence of its interaction with the cofactor within the prothrombinase complex.

Characterization of a barotolerant mutant of the yeast Saccharomyces cerevisiae: importance of trehalose content and membrane fluidity

A barotolerant mutant of Saccharomyces cerevisiae and its parent were compared with respect to the barotolerance of each strain, intracellular content of trehalose, amounts of three major heat shock proteins (hsps), relative proportion of unsaturated and saturated fatty acids in membrane phospholipids, and membrane fluidity. Changes in the amount of trehalose, but not hsps, were associated with changes in the barotolerance of each strain during the different phases of growth and with the difference in barotolerance between the mutant and parent strain. The membrane fluidity in the case of the mutant was low, while that of the parent was high. These results suggest that accumulation of trehalose and the condition of the membrane are more important for barotolerance than accumulation of hsps.

Characterization of a barotolerant mutant of the yeast Saccharomyces cerevisiae: importance of trehalose content and membrane fluidity.

A barotolerant mutant of Saccharomyces cerevisiae and its parent were compared with respect to the barotolerance of each strain, intracellular content of trehalose, amounts of three major heat shock proteins (hsps), relative proportion of unsaturated and saturated fatty acids in membrane phospholipids, and membrane fluidity. Changes in the amount of trehalose, but not hsps, were associated with changes in the barotolerance of each strain during the different phases of growth and with the difference in barotolerance between the mutant and parent strain. The membrane fluidity in the case of the mutant was low, while that of the parent was high. These results suggest that accumulation of trehalose and the condition of the membrane are more important for barotolerance than accumulation of hsps.

Evaluation of fatty acids in membrane phospholipids of erythrocytes in retinitis pigmentosa patients.

Several investigators have observed abnormalities of plasma DHA in various common forms of retinitis pigmentosa. Erythrocyte membranes are an accessible tissue which may more closely reflect neural membrane fatty acid levels, but little data is yet available on their fatty acid composition in retinitis pigmentosa patients. We have evaluated the DHA levels in red blood cells membrane phospholipids in various genetic forms of retinitis pigmentosa and relative controls. The results obtained indicate significantly lower DHA levels in red blood cell membrane phospholipids in retinitis pigmentosa patients with respect to controls (p < 0.01). This data is significant for autosomal dominant forms only (p < 0.01). Fatty acid content alterations in membrane phospholipids of red blood cells may be considered as markers for abnormalities in the lipid metabolism which disturb the retina integrity.

Clinical Use of Fatty Acid Supplements in Dogs

Abstract— Fatty acids in membrane phospholipids are metabolized by cellular enzymes into a variety of eicosanoids that function in the regulation of normal physiology and serve as mediators of inflammation. Fatty acid supplements can alter the types of eicosanoids produced. Many investigations using fatty acids in the treatment of nutritional deficiencies, seborrhea, atopic dermatitis, cardiovascular, neoplastic, ocular and musculoskeletal diseases of dogs have been conducted over the past decade. Additional studies are needed to determine the optimal levels of linoleic acid, gamma‐linolenic acid and/or eicosapentaneoic acid in the diets of dogs for the maintenance of normal homeostasis and for the treatment of inflammatory, degenerative and neoplastic diseases.

Effects of sphingosine on peripheral membrane interactions: Comparison of adriamycin, cytochrome c, and phospholipase A2

As revealed by resonance energy transfer utilizing pyrene-labeled phosphatidylcholine donor, the mainly electrostatically controlled binding of adriamycin (Adr) and cytochrome c (cyt c) to mixed egg yolk phosphatidic acid/phosphatidylcholine (eggPA/eggPC, 15:85 molar ratio) liposomes was reversed upon the inclusion of increasing contents of sphingosine. At a [sphingosine]/[eggPA] molar ratio of approximately 2:1, the degree of fluorescence quenching by cyt c and Adr was approximately the same as when using liposomes lacking eggPA. Similarly, the increase in the surface pressure of sphingosine/eggPA monolayers on an air/water interface due to the membrane penetration of either cyt c or Adr was progressively reduced by increasing the content of sphingosine in the monolayers. The above critical [sphingosine]/[acidic phospholipid] stoichiometry yielding dissociation of the positively charged ligands Adr or cyt c from membrane acidic phospholipids was shifted from 2:1 to 1:1 upon substituting egg phosphatidylglycerol (eggPG) for eggPA. Accordingly, charge neutralization of the acidic phospholipids by sphingosine could be involved. One eggPA (having maximally two negative charges) appears to require two molecules of sphingosine whereas the maximally singly charged eggPG is neutralized by one sphingosine. For comparison we also studied the effects of sphingosine on the phospholipase A2 catalyzed hydrolysis of the pyrene-labeled acidic alkylacyl phospholipid analog 1-octacosanyl-2-[6-(pyren-1-yl)]hexanoyl-sn-glycero-3- phosphatidylmethanol (C28-O-PHPM) and the corresponding phosphatidylcholine (C28-O-PHPC). In the presence of low Ca2+ concentrations (approximately 50 nM) limiting the rate of the enzymatic reaction, sphingosine gradually inhibited the hydrolysis of phosphatidylcholine, and at 1:6 sphingosine/C28-O-PHPC a nearly complete lack of hydrolysis was evident.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-specific hole formation in the Escherichia coli inner membrane by λ S proteins is independent of cellular secY and secA functions and of the proportion of membrane acidic phospholipids

Formation of the lesion in the Escherichia coli inner membrane caused by lambda lysis protein S was examined by electron microscopy. We also show that macromolecules exceeding the size of the lambda R transglycosylase can pass through the S-dependent hole and that assembly of the S-dependent hole is independent of the proportion of acidic phospholipids in the inner membrane and of components of the cellular transport machinery.

Non-specific hole formation in the Escherichia coli inner membrane by λ S proteins is independent of cellular secY and secA functions and of the proportion of membrane acidic phospholipids

Formation of the lesion in the Escherichia coli inner membrane caused by λ lysis protein S was examined by electron microscopy. We also show that macromolecules exceeding the size of the λ R transglycosylase can pass through the S-dependent hole and that assembly of the S-dependent hole is independent of the proportion of acidic phospholipids in the inner membrane and of components of the cellular transport machinery.

Inhibition of vasopressin-induced formation of diradylglycerols in vascular smooth muscle cells by incorporation of eicosapentaenoic acid in membrane phospholipids.

Eicosapentaenoic acid and linoleic acid exert antihypertensive effects by an unknown mechanism unrelated to prostanoids, a property which is not shared by arachidonic acid. This study investigated the influence of these three acids on the formation of diradylglycerols and phosphatidic acid, key intracellular messengers involved in the mediation of agonist-induced vascular smooth muscle cell contraction. Rat mesenteric artery vascular smooth muscle cells in culture were pre-incubated for 24 h with eicosapentaenoic acid, linoleic acid or arachidonic acid. After thorough washing the cells were then incubated for 20 min in the presence of arginine vasopressin or vehicle, either immediately or following cell labelling with 32P-orthophosphate. The fatty acid composition of cell lipids was determined by gas chromatography after transesterification in the presence of boron trifluoride and methanol. Diradylglycerols and 32P-phosphatidic acid were purified from cell lipid extracts by thin-layer chromatography and diradylglycerols were analysed. Incubation of vascular smooth muscle cells with eicosapentaenoic acid, linoleic acid or arachidonic acid resulted in the incorporation of these fatty acids at the sn-2 position of membrane phospholipids, mainly phosphatidylcholine and phosphatidylethanolamine. Eicosapentaenoic acid treatment was associated with a reduction, and linoleic acid treatment with an increase in the relative proportions of arachidonic acid found in cell phospholipids. Arginine vasopressin stimulated the formation of both diradylglycerols and 32P-phosphatidic acid. The arginine vasopressin-induced stimulation of diradylglycerols accumulation was almost completely abolished in eicosapentaenoic acid-treated cells, whereas it was not modified by linoleic acid or by arachidonic acid treatment. The arginine vasopressin-stimulated formation of 32P-phosphatidic acid was significantly inhibited by linoleic acid treatment but was not influenced by eicosapentaenoic acid or arachidonic acid treatment. The incorporation of eicosapentaenoic acid or linoleic acid at the sn-2 position of membrane phospholipids leads to an inhibition of arginine vasopressin-induced formation of diradylglycerols or phosphatidic acid, respectively, in rat mesenteric artery vascular smooth muscle cells in culture. These properties may contribute to the antihypertensive effects in these fatty acids in vitro.

Adaptational Changes of Fatty Acid Composition and the Physical State of Membrane Lipids Following the Change of Growth Temperature in Yersinia enterocolitica

Yersinia enterocolitica is capable of growing in a broad range of temperatures from 4 to 45 C. How this organism alters its membrane lipids in response to the change of growth temperature is very interesting. The fatty acids of membrane lipids of cells cultured at 5, 15, 25 and 37 C were analyzed and the physical states of these membrane lipids were characterized. The major phospholipids of this bacterium were phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, lysophosphatidylglycerol and lysophosphatidylethanolamine. No significant difference in phospholipid composition in response to culture temperatures was observed. It was reported in our previous paper that the major fatty acids of membrane phospholipids of Y. enterocolitica were C15:0, C16:0, C16:1, cyclopropane C17:0 and C18:0. Some differences in the fatty acid composition were, however, observed with the change of culture temperature. When the culture temperature was raised, the saturated and cyclopropane fatty acids substantially increased and the unsaturated ones decreased. A reverse phenomenon was observed when culture temperature was lowered. From the viewpoints of membrane physical state, adaptational changes were analyzed using a nylon microcapsule method. Phase transition in membrane lipids of cells grown at each culture temperature took place in the range of about 5 C below and about 10 C above the culture temperature. It is, therefore, considered that Y. enterocolitica maintains its membrane rigidity and fluidity in response to growth temperature by changing the membrane fatty acid composition.

Inhibition of the action of a stimulatory GDP/GTP exchange protein for smg p21 by acidic membrane phospholipids.

A stimulatory GDP/GTP exchange protein for smg p21 (smg p21 GDS) stimulated the dissociation of GDP from smg p21B. This reaction was inhibited by acidic membrane phospholipids such as phosphatidylinositol, phosphatidylinositol‐4‐monophosphate, phosphatidylinositol‐4‐,5‐bisphosphate, phosphatidic acid, and phosphatidylserine but not by phosphatidykholine or phosphatidylethanolamine. These acidic phospholipids inhibited the smg p21 GDS action in a manner competitive with both smg p21 GDS and smg p21B. smg p21 GDS has other actions to inhibit the binding of smg p21B to membranes and to induce the dissociation of prebound smg p21B from the membranes. The acidic phospholipids also inhibited these two actions of smg p21 GDS. smgp21B has a polybasic region and an isoprenoid moiety in its C‐terminal region which are necessary for its membrane‐binding activity and its sensitivity to the smg p21 GDS actions. Therefore, it is possible that acidic membrane phospholipids interact with this polybasic region and thereby inhibit the smg p21 GDS actions.

The smg GDS-induced activation of smg p21 is initiated by cyclic AMP-dependent protein kinase-catalyzed phosphorylation of smg p21

We have shown that smg p21B is phosphorylated by cyclic AMP-dependent protein kinase (protein kinase A) and that membrane acidic phospholipids such as phosphatidic acid and phosphatidylinositol markedly inhibit the smg GDS-induced activation of smg p21B. However, we show here that phosphatidic acid and phosphatidylinositol exhibit a less inhibitory effect on the smg GDS-induced activation of the phosphorylated form of smg p21B. Thus, in the presence of membrane acidic phospholipids, the smg GDS-induced activation of smg p21B is totally dependent on the protein kinase A-catalyzed phosphorylation of smg p21B. Since smg p21B is located on the membranes in resting cells, it is likely that the smg GDS-induced activation of smg p21B is initiated by the protein kinase A activation.

In vitro andin vivo responses of rat tissues to cadmium-induced lipid peroxidation

Oxidative destruction of polyunsaturated fatty acids of membrane phospholipids, a phenomenon generally termed lipid peroxidation (LPO), is considered to be an important mechanism of toxicity for a wide variety of chemicals. Among these, cadmium (Cd), a pollutant of industrial and environmental importance, induces LPO in various tissues despite its apparent inability to directly generate free radicals under physiological conditions. Consequently, although LPO is not the primary mechanism of Cd toxicity, it represents an early intracellular response of tissues following exposure to Cd compounds. Recently, the authors reported the in vitro specific response to LPO of liver, lung, heart, kidney, testes and brain tissues incubated with various concentrations of CdCl{sub 2}. LPO was assessed by the measurement of thiobarbituric acid reactive substances (TBARS) which include malondialdehyde and lipid hydroperoxydes, and by gaz chromatographic analysis of evolved hydrocarbons, namely ethane and pentane. To compare the results obtained by both methods, they standardized TEP and TBARS values against incubated controls after subtracting endogenous levels of TBARS (time 0 values) because the measurement of endogenous levels of TEP in tissue incubates is not practicable. The objective of the present report is to clarify this concept in order to adequately compare the data obtained frommore » in vitro and in vivo studies. This should permit a better assessment of the relative importance of tissue responses to LPO following exposure to Cd.« less

Fish Oil Fatty Acids and Experimental Arthritis

Maintenance of mice on dietary regimens containing fish oil decreases severity of collagen-induced arthritis. Macrophages from fish oil fed animals had decreased omega-6 and significant amounts of omega-3 polyunsaturated fatty acids in membrane phospholipids and produced significantly less prostaglandins than macrophages from corn oil fed animals. Gender differences in both prostaglandin production and susceptibility to arthritis were noted.

The decrease in osmotic fragility of erythrocytes during supplementation with n-3 fatty acids is a transient phenomenon

In order to study the effect of dietary n-3 fatty acids on osmotic fragility of human erythrocytes, 11 healthy subjects were given a supplement of 6 g day-1 of an oil containing 50% n-3 fatty acids for either 14 days or 34 days. Fourteen days after start the osmotic fragility was decreased by 60-80% (buffer-salt concentration 0.41%), and the level of n-3 fatty acids in membrane phospholipids was increased by approximately 60%. The decrease in fragility was less marked after 24 days and almost at the pre-supplementation level after 34 days. There was no correlation between changes in fragility and in the fatty acid pattern of membrane phospholipids of the erythrocytes. The changes induced in fatty acid composition of phospholipids did not affect membrane fluidity. It is concluded that factors other than the nature of fatty acids in membranes may be involved in modifying osmotic fragility, and that there is no correlation between membrane fragility and membrane fluidity.

Influence of dietary linseed oil and sunflower seed oil on some mechanical and metabolic parameters of isolated working rat hearts

The role played by membrane lipid environment on cardiac function remains poorly defined. The polyunsaturated fatty acid profile of myocardial phospholipids could be of utmost importance in the regulation of key-enzyme activities. This study was undertaken to determine whether selective incorporation of n-6 or n-3 fatty acids in membrane phospholipids might influence cardiac mechanical performances and metabolism. For 8 wk, male weaning Wistar rats were fed a semi-purified diet containing either 10% sunflower seed oil (72% C18:2 n-6) or 10% linseed oil (54% C18:3 n-3) as the sole source of lipids. The hearts were then removed and perfused according to working mode with a Krebs-Henseleit buffer containing glucose (11 mM) and insulin (10 Ul/l). Cardiac rate, coronary and aortic flows and ejection fraction were monitored after 30 min of perfusion. Myocardial metabolism was estimated by evaluating the intracellular fate of 1-14C palmitate. Sunflower seed oil and linseed oil feeding did not modify either coronary or aortic flow, which suggests that cardiac mechanical work was not affected by the diets. Conversely, cardiac rate was significantly decreased (-18%; P less than 0.01) when rats were fed the n-3 polyunsaturated fatty acid rich diet. Radioanalysis of the myocardial metabolism suggested that replacing n-6 polyunsaturated fatty acids by n-3 polyunsaturated fatty acids: i) did not alter palmitate uptake; ii) prolonged palmitate incorporation into cardiac triglycerides; iii) reduced beta-oxidation of palmitic acid. These results support the assumption that dietary fatty acids, particularly n-6 and n-3 fatty acids, play an important role in the regulation of cardiac mechanical and metabolic activity.

ACIDIC PHOSPHOLIPIDS IN SYNAPTOSOMAL PLASMA MEMBRANES DURING REPEATED EPISODES OF PHYSICAL ETHANOL DEPENDENCE IN THE RAT

The effects of repeated episodes of ethanol intoxication and the hyperexcitable state of withdrawal on the synaptosomal concentration of acidic phospholipids were studied in rats. There was no indication that cumulative changes in the synaptosomal acidic phospholipid composition in general occurred during multiple episodes of ethanol intoxication and withdrawal. There was, however, a statistically significant decrease in acidic phospholipid concentration (phosphatidylinositol; PI) in synaptosomal membranes from animals revealing spontaneous convulsive behaviour during ethanol withdrawal. Hypothetically this may reflect an inability to increase acidic phospholipid membrane content and thus to adaptively increase the seizure threshold during withdrawal.

Preferential uptake and metabolism of docosahexaenoic acid in membrane phospholipids from rod and cone photoreceptor cells of human and monkey retinas

The uptake, metabolism, and cellular distribution of [3H]docosahexaenoic acid (DHA) in human and monkey retinas were studied with biochemical and autoradiographic techniques. In specimens from two human retina biopsies, incubated for 4 hr or 6 hr with [3H]docosahexaenoic acid (110 nM), 80% of the esterified [3H]fatty acid was recovered in phospholipids and the remainder in triacylglycerols and diacylglycerols. The distribution of [3H]DHA in individual phospholipids (PL) was similar in both retinas, with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) accounting for most of the label. A similar labeling profile was observed in glycerolipids from monkey retina, and after 1 hr of incubation, high labeling of phosphatidic acid (PA, 11%) and phosphatidylinositol (PI, 20%) was observed. In both human and monkey retinas, a preferential uptake of [3H]DHA by photoreceptor cells was revealed by autoradiography. Cone photoreceptors showed a slightly higher density of silver grains in their inner segments than did rod photoreceptors. Photoreceptors accounted for 59% and 79% of the total [3H]DHA taken up by the human and monkey retinas, respectively, the remainder being distributed throughout the neural retina. In conclusion, this study shows for the first time that in human and monkey retinas, DHA is taken up with a high degree of selectivity by photoreceptor cells, and then becomes esterified mainly into phospholipids that will be subsequently utilized for the synthesis of new disc membranes.

Effect of In Vivo Modulation of Membrane Docosahexaenoic Acid Levels on the Dopamine‐Dependent Adenylate Cyclase Activity in the Rat Retina

We have studied the effect of a dietary deprivation of n-3 fatty acids on the activity of the dopamine (DA)-dependent adenylate cyclase in the rat retina. Experiments were conducted in 6-month-old rats raised on semipurified diets containing either safflower oil (n-3 deficient diet) or soybean oil (control diet). The levels of docosahexaenoic acid [22:6 (n-3)] in retinal phospholipids were significantly decreased in n-3 deficient rats (35-42% of control levels). This was compensated by a rise in 22:5 (n-6), the total content of polyunsaturated fatty acids (PUFA) remaining approximately constant. Adenylate cyclase activity was measured in retinal membrane preparations from dark-adapted or light-exposed rats. The enzyme activity was stimulated by DA and SKF 38393 in a light-dependent fashion. The activation was lower in rats exposed to light than in dark-adapted animals, suggesting a down-regulation of the D1 DA receptors by light. The activation by guanine nucleotides and forskolin was also decreased in light-exposed rats. There was no significant effect of the dietary regimen on the various adenylate cyclase activities and their response to light. Furthermore, the guanine nucleotide- and DA-dependent adenylate cyclase activities of retinal membranes were found to be relatively resistant to changes in membrane fluidity induced in vitro by benzyl alcohol. The results indicate that in the absence of changes in total PUFA content, a decreased ratio of n-3 to n-6 fatty acids in membrane phospholipids does not significantly affect the properties of adenylate cyclase in the rat retina.