Phospholipid composition of human, dog and guinea pig adrenal cortex

The phospholipid composition was studied in human and dog adrenal cortex and in guinea pig adrenal tissue. The major phospholipids of adrenal cortex were phosphatidylcholine and phosphatidylethanolamine whose ratio in the human, dog and guinea pig tissues was 2.16, 2.01, 1.61, respectively. Phosphatidylinositol, phosphatidylserine, sphingomyelin, diphosphatidylglycerin, lysophosphatidylcholine and lysophosphatidyl-ethanolamine were also found in adrenal cortex. A quantitative phospholipid composition of the human adrenal cortex was close to the dog one. The fatty acid composition of phosphatidylcholine from human and dog adrenal cortex was determined and some differences were shown.     

Lipid composition of miniature pig platelets

1. Analyses of platelet lipid composition were carried out on material pooled from male and female miniature pigs. 2. The cholesterol/phospholipid molar ratio was 0.6. 3. Phosphatidylcholine represents the major class of phospholipids (47%) and phosphatidylinositol the minor (2%). 4. The main fatty acids of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and sphingomyelin were: palmitic, stearic, oleic, linoleic and arachidonic acids. 5. The ratios of saturated to unsaturated fatty acids were: sphingomyelin, 1.7; phosphatidylcholine, 1.2; phosphatidylserine, 0.9; phosphatidylethanolamine and phosphatidylinositol, 0.6. 6. Our results suggests that human and miniature pig platelet lipids bear several characteristics in common. This fact would allow miniature pig to be used as a new experimental model.     

Membrane-damaging action of staphylococcal alpha-toxin on phospholipid-cholesterol liposomes

The mechanism of membrane damage by staphylococcal alpha-toxin was studied using carboxyfluorescein (internal marker)-loaded multilamellar liposomes prepared from various phospholipids and cholesterol. Liposomes composed of phosphatidylcholine or sphingomyelin and cholesterol bound alpha-toxin and released carboxyfluorescein in a dose dependent manner, when they were exposed to alpha-toxin of concentrations higher than 1 or 8 micrograms/ml, respectively. In contrast, the other liposomes composed of phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol or phosphatidylinositol plus cholesterol were not susceptible to the toxin even at high concentrations up to 870 micrograms/ml. The insensitive liposomes containing either phosphatidylserine or phosphatidylglycerol were made sensitive to alpha-toxin by inserting phosphatidylcholine into the liposomal membranes. In addition, phosphorylcholine inhibited the toxin-induced marker release from liposomes. These results indicated that the choline-containing phospholipids are required for the interaction between alpha-toxin and liposomal membranes. Susceptibility of liposomes containing phosphatidylcholine or sphingomyelin increased with the increase in cholesterol contents of the liposomes. Based on these results, we propose that the choline-containing phospholipids are possible membrane components or structures responsible for the toxin-membrane interaction, which leads to damage of membranes. Furthermore, cholesterol may facilitate the interaction between alpha-toxin and membrane as a structural component of the membrane.     

Effect of phosphatidylinositol replacement by diacylglycerol on various physical properties of artificial membranes with respect to the role of phosphatidylinositol response

In an attempt to gain insight into the physiological role of phosphatidylinositol turnover enhanced by extracellular stimuli, the physical properties of artificial membranes (egg yolk phosphatidylcholine/bovine brain phosphatidylserine) containing phosphatidylinositol or diacylglycerol were studied by ESR using spin probes and freeze-fracture electron microscopy. Diacylglycerol lost both the ability to form lipid bilayer structures and its susceptibility to calcium ions. Yeast phosphatidylinositol included in dipalmitoylphosphatidylcholine liposomes lowered the phase transition temperature of dipalmitoylphosphatidylcholine and expanded the temperature range of phase transition. However, diacylglycerol at the same concentration did not undergo the effects caused by phosphatidylinositol but the phase transition temperature was slightly raised. Phase separation of phosphatidylserine induced by calcium ions was enhanced when the phosphatidylinositol was replaced by diacylglycerol in phosphatidylcholine/ phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) mixtures. The mobility of phosphatidylcholine spin probe was decreased in phosphatidylcholine/ phosphatidylserine/diacylglycerol (3:5:2, by molar ratio) liposomes compared with phosphatidylcholine/phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) liposomes. An additional component from protonated stearic acid spin probes was observed in phosphatidylcholine/phosphatidylinositol (8:2, by molar ratio) liposomes at 40°C, whereas the component was not seen in phosphatidylcholine/diacylglycerol (8:2, by molar ratio) liposomes. This may indicate the alteration of surface charge induced by the replacement of phosphatidylinositol by diacylglycerol. Indeed, in the presence of 1 mM Ca²⁺, the additional component was removed by an electrostatic interaction between Ca²⁺ and phosphatidylinositol molecules in phosphatidylcholine/phosphatidylinositol liposomes at 40°C. These results support the hypothesis that the enhanced turnover of phosphatidylinositol may play a triggering role for various cellular responses to exogenous stimuli by altering membrane physical states.     

Interaction of bovine brain phospholipid exchange protein with liposomes of different lipid composition

The major phospholipid exchange protein from bovine brain catalyzes the transfer of phosphatidylinositol and phosphatidylcholine between rat liver microsomes and sonicated liposomes. The effect of liposomal lipid composition on the transfer of these phospholipids has been investigated. Standard liposomes contained phosphatidylcholine-phosphatidic acid (98:2, mol%); in general, phosphatidylcholine was substituted by various positively charged, negatively charged, or zwitterionic lipids. The transfer of phosphatidylinositol was essentially unaffected by the incorporation into liposomes of phosphatidic acid, phosphatidylserine, or phosphatidylglycerol (5–20 mol%) but strongly depressed by the incorporation of stearylamine (10–40 mol%). Marked stimulation (2–4-fold) of transfer activity was observed into liposomes containing phosphatidylethanolamine (2–40 mol%). The inclusion of sphingomyelin in the acceptor liposomes gave mixed results: stimulation at low levels (2–10 mol%) and inhibition at higher levels (up to 40 mol%). Cholesterol slightly diminished transfer activity at a liposome cholesterol/phospholipid molar ratio of 0.81. Similar effects were noted for the transfer to phosphatidylcholine from microsomes to these various liposomes. Compared to standard liposomes, the magnitude of $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ tended to increase for liposomes which depressed phospholipid transfer and to decrease for those which stimulated; little change was observed in the values of $\text{V}$. Single phospholipid liposomes of phosphatidylinositol were inhibitory when added to standard liposomes.     

Phospholipids of Entamoeba invadens.

The major phosphoglycerides present in Entamoeba invadens are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol. Furthermore, three different sphingolipids could be isolated from the amoeba. In addition to sphingomyelin and a phosphonolipid, ceramide phosphonylethanolamine, a previously unknown sphingolipid was present. This sphingolipid contained a long chain base, inositol, and phosphorus in the ratio of 0.97:0.97: 1.0 and could be identified as ceramide phosphorylinositol. The various individual phospholipids showed different rates of turnover. Phosphatidic acid and phosphatidylinositol had, relative to the other phospholipids, a short half-time of about 12 h. Phosphatidylethanolamine and ceramide phosphorylinositol had a half-time of about 24 and 30 h, respectively. The major phospholipid, phosphatidylcholine and also sphingomyelin and phosphatidylserine showed no turnover. In contrast to the phosphoglycerides, the sphingolipid composition of the amoeba cultivated in different media was rather variable, while the total sphingolipid content remained at 21% of the total amount of phospholipids. The amount of ceramide phosphorylinositol was almost doubled in the cells cultivated on the serum-free medium (T), whereas the amount of sphingomyelin and ceramide phosphonylethanolamine decreased. Evidence is presented that these alterations in the sphingolipid composition of E. invadens are related to the amount of unsaturated fatty acids which were present in the culture medium.     

Effect of phosphatidylinositol replacement by diacylglycerol on various physical properties of artificial membranes with respect to the role of phosphatidylinositol response

In an attempt to gain insight into the physiological role of phosphatidylinositol turnover enhanced by extracellular stimuli, the physical properties of artificial membranes (egg yolk phosphatidylcholine/bovine brain phosphatidylserine) containing phosphatidylinositol or diacylglycerol were studied by ESR using spin probes and freeze-fracture electron microscopy. Diacylglycerol lost both the ability to form lipid bilayer structures and its susceptibility to calcium ions. Yeast phosphatidylinositol included in dipalmitoylphosphatidylcholine liposomes lowered the phase transition temperature of dipalmitoylphosphatidylcholine and expanded the temperature range of phase transition. However, diacylglycerol at the same concentration did not undergo the effects caused by phosphatidylinositol but the phase transition temperature was slightly raised. Phase separation of phosphatidylserine induced by calcium ions was enhanced when the phosphatidylinositol was replaced by diacylglycerol in phosphatidylcholine/phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) mixtures. The mobility of phosphatidylcholine spin probe was decreased in phosphatidylcholine/phosphatidylserine/diacylglycerol (3:5:2, by molar ratio) liposomes compared with phosphatidylcholine/phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) liposomes. An additional component from protonated stearic acid spin probes was observed in phosphatidylcholine/phosphatidylinositol (8:2, by molar ratio) liposomes at 40 degrees C, whereas the component was not seen in phosphatidylcholine/diacylglycerol (8:2, by molar ratio) liposomes. This may indicate the alteration of surface charge induced by the replacement of phosphatidylinositol by diacylglycerol. Indeed, in the presence of 1 mM Ca2+, the additional component was removed by an electrostatic interaction between Ca2+ and phosphatidylinositol molecules in phosphatidylcholine/phosphatidylinositol liposomes at 40 degrees C. These results support the hypothesis that the enhanced turnover of phosphatidylinositol may play a triggering role for various cellular responses to exogenous stimuli by altering membrane physical states.     

Effect of retinoic acid on the acylation of phospholipids in granulocytes in vitro

The influence of retinoic acid on the incorporation of [1-14C]palmitic acid and [1-14C]arachidonic acid into phospholipids was examined in guinea pig peritoneal granulocytes. All-trans-retinoic acid inhibited the incorporation of both fatty acids into phosphatidic acid and phosphatidylinositol. However, it stimulated the incorporation of both fatty acids into phosphatidylcholine but not other phospholipids. All-trans-retinoic acid was more effective than 13-cis-retinoic acid. The influence of all-trans-retinoic acid on the acylation of phospholipids was concentration-dependent with significant effect occurring at 2.1 microM. The loss of labeled fatty acids from prelabeled phospholipids and the transport of labeled fatty acids into granulocytes were not responsive to the presence of retinoic acid in the incubation media. These results suggest that retinoic acid may affect the activities of acyltransferases involved in the synthesis of phosphatidic acid, phosphatidylinositol and phosphatidylcholine.     

Lipid composition of Trypanosoma cruzi.

1. Lipid composition of Trypanosoma cruzi epimastigote form in culture consist of 35% of phospholipids and 65% of neutral lipids. 2. Among the phospholipids, phosphatidylcholine is the more abundant (44%), followed by phosphatidylethanolamine (28%), phosphatidylinositol (12%), sphingomyelin (4%), and smaller amounts of cardiolipin, phosphatidic acid, lysolecithin, phosphatidylserine (traces), and an unidentified phospholipid (3%). 3. Pulse labeling with 32P showed highest specific incorporation in phosphatidylethanolamine, followed by phosphatidylinositol and phosphatidylcholine, suggesting a more active role for phosphatidylethanolamine in these organisms.     

Asymmetry of the phospholipid bilayer of rat liver endoplasmic reticulum

The phospholipids of intact microsomal membranes were hydrolysed 50% by phospholipase C of Clostridium welchii, without loss of the secretory protein contents of the vesicle, which are therefore not permeable to the phospholipase. Phospholipids extracted from microsomes and dispersed by sonication were hydrolysed rapidly by phospholipase C-Cl. welchii with the exception of phosphatidylinositol. Assuming that only the phospholipids of the outside of the bilayer of the microsomal membrane are hydrolysed in intact vesicles, the composition of this leaflet was calculated as 84% phosphatidylcholine, 8% phosphatidylethanolamine, 9% sphingomyelin and 4% phosphatidylserine, and that of the inner leaflet 28% phosphatidylcholine, 37% phosphatidylethanolamine, 6% phosphatidylserine and 5% sphingomyelin. Microsomal vesicles were opened and their contents released in part by incubation with deoxycholate (0.098%) lysophosphatidylcholine (0.005%) or treatment with the French pressure cell. Under these conditions, hydrolysis of the phospholipids by phospholipase C-Cl. welchii was increased and this was mainly due to increased hydrolysis of those phospholipids assigned to the inner leaflet of the bilayer, phosphatidylethanolamine and phosphatidylserine. Phospholipase A₂ of bee venom and phospholipase C of Bacillus cereus caused rapid loss of vesicle contents and complete hydrolysis of the membrane phospholipids, with the exception of sphingomyelin which is not hydrolysed by the former enzyme.     

Effect of endurance training on the phospholipid content of skeletal muscles in the rat

Only few data are available on the effect of training on phospholipid metabolism in skeletal muscles. The aim of the present study was to examine the effect of 6 weeks of endurance training on the content of particular phospholipid fractions and on the incorporation of blood-borne [14C]-palmitic acid into the phospholipids in different skeletal muscles (white and red sections of the gastrocnemius, the soleus and the diaphragm) of the rat. Lipids were extracted from the muscles and separated using thin-layer chromatography into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, cardiolipin and neutral lipids (this fraction being composed mostly of triacylglycerols). It was found that training did not affect the content of any phospholipid fraction in soleus muscle. It increased the content of sphingomyelin in white gastrocnemius muscle, cardiolipin and phosphatidylethanolamine in red gastrocnemius muscle and phosphatidylinositol in white gastrocnemius muscle and diaphragm. The total phospholipid content in red gastrocnemius muscle of the trained group was higher than in the control group. Training reduced the specific activity of sphingomyelin and cardiolipin in all muscles, phosphatidylcholine in soleus, red, and white gastrocnemius muscles, phosphatidylserine in all muscles, phosphatidylinositol in all except the soleus muscle, and phosphatidylethanolamine in hindleg muscles, but not in the diaphragm compared to the corresponding values in the sedentary group. It was concluded that endurance training affects skeletal muscle phospholipid content and the rate of incorporation of the blood-borne [14C]palmitic acid into the phospholipid moieties.     

24-Hour monitoring of the serum lipid profile in normal and obese women

In fast and in fat-load elevated the free cholesterol level and decreased the total phospholipids contents in the blood serum of both normal and obese women. Subsequently, normal women revealed a drop in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, whereas in obese women the phosphatidylcholine level remained stable during the test. Contents of phosphatidylcholine, phosphatic acid and glycerophosphate was still lowering in 24 hours after the fat load.     

Effect of triiodothyronine on the content of phospholipids in the rat liver nuclei.

The aim of the present study was to examine the effect of triiodothyronine (T3) on the content of phospholipids and on the incorporation of blood-borne palmitic acid into the phospholipid moieties in the nuclei of the rat liver. T3 was administered daily for 7 days, 10 microg x 100 g(-1). The control rats were treated with saline. Each rat received 14C-palmitic acid, intravenously suspended in serum. 30 min after administration of the label, samples of the liver were taken. The nuclei were isolated in sucrose gradient. Phospholipids were extracted from the nuclei fraction and from the liver homogenate. They were separated into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and cardiolipin. The content and radioactivity of each fraction was measured. It was found that treatment with T3 reduced the content of phosphatidylinositol and increased the content of cardiolipin in the nuclear fraction. In the liver homogenate, the content of phosphatidylinositol decreased and the content of phosphatidylethanolamine and cardiolipin increased after treatment with T3. The total content of phospholipids after treatment with T3 remained unchanged, both in the nuclear fraction and in the liver homogenate. T3 reduced the specific activity of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and cardiolipin and had no effect on the specific activity of sphingomyelin and phosphatidylinositol both in the fraction of the nuclei and the liver homogenate. It is concluded that excess of triiodothyronine affects the content of phospholipids in the nuclei. The changes in the content of phospholipids in the nuclei largely reflect changes in their content in the liver.     

Acidic phospholipids, unsaturated fatty acids, and limited proteolysis mimic the effect of calmodulin on the purified erythrocyte Ca2+ - ATPase

The purified Ca2+-pumping ATPase of human erythrocyte membranes (Niggli, V., Adunyah, E. S., Penniston, J. T., and Carafoli, E. (1981) J. Biol. Chem. 256, 395-401) can be stimulated, in the absence of calmodulin, by other treatments. 1. A variety of acidic phospholipids (phosphatidylserine, cardiolipin, phosphatidylinositol, and phosphatidic acid) stimulate the Vmax and decrease the Km (Ca2+) of the isolated enzyme to the same extent as calmodulin. Unsaturated fatty acids (oleic and linoleic acid) have the same effect as phospholipids but at lower concentrations. Neutral phospholipids (phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine) have no effect on the enzyme. The minimal proportion of acidic phospholipids in the environment of the enzyme necessary for full stimulation is about 40%. 2. The isolated enzyme, after reconstitution in phosphatidylcholine liposomes in the absence of calmodulin, can be activated by limited proteolysis. The trypsinized enzyme has the same high Vmax and high affinity for Ca2+ of the enzyme in the presence of calmodulin.     

THE EXCHANGE OF PHOSPHATIDYLCHOLINE AND OF PHOSPHATIDYLINOSITOL IN SHEEP BRAIN

—The exchange of phospholipids between liposomes and brain mitochondria has been studied in the presence of pH 5·1 supernatant fluids derived from rat, guinea pig, sheep and ox brains. The exchange phenomenon was similar to that observed in liver and heart, but phosphatidylinositol and not phosphatidylcholine was the most rapidly exchanging phospholipid. The phosphatidylcholine exchange activity was purified 186-fold from sheep brain and the protein fraction contained two major and several minor protein species. The phosphatidylcholine and phosphatidylinositol exchange activities have been shown to have very similar molecular weights and isoelectric points. However, their behaviour in response to changes in liposomal surface charge suggested that separate proteins might be involved in stimulating the exchange of the two phospholipid classes.     

Effect of phospholipase C treatment on the activity of the particulate cyclic nucleotide phosphodiesterase of rat brain

1. The activity of cAMP phosphodiesterase (PDE) was studied in a 10,000 g particulate fraction prepared from rat brain. 2. Phospholipase C such as sphingomyelin choline phosphodiesterase (SMase), phosphatidylinositol phosphodiesterase (PIase) and phosphatidylcholine phosphohydrolase (PCase) were used to deplete phospholipid(s) from the particulate fraction and their effects on PDE activity were investigated. 3. Treatment with SMase or PIase did not affect PDE activity whereas treatment with PCase resulted in inhibition. 4. It was also found that the PCase used not only hydrolyzed phosphatidylcholine but also other phospholipids such as phosphatidylethanolamine, phosphatidylserine and sphingomyelin.     

Fatty acid composition and dynamics of phospholipids from hake (Merluccius hubbsi) spinal cord and brain and sea bass (Acanthustius brasilianus) brain.

This work studies the phospholipid and fatty acid composition in hake brain and spinal cord and in sea bass brain. Fluorescence anisotropy of phospholipid vesicles labeled with 1,6-diphenyl hexatriene was measured to investigate the associated dynamic properties. In all tissues studied, phosphatidylcholine and phosphatidylethanolamine were the major constituents with minor contributions of phosphatidylserine, phosphatidylinositol and sphingomyelin. Fatty acids belong to the n-9 and n-3 series exclusively. Phosphatidylinositol from hake spinal cord and phosphatidylethanolamine and phosphatidylserine from hake brain contain the greatest percentages of eicosa-5,8,11,14,17-pentaenoic (20:5) and docosa-4,7,10,13,16,19-hexaenoic (22:6), respectively. For all fractions studied the total content of saturated fatty acids increases in the order of hake spinal cord, hake brain, sea bass brain together with a decrease in the sum of monounsaturated fatty acids. The comparison between fluorescence anisotropy values and fatty acid composition clearly demonstrates that saturated acids and 20:5 and 22:6 exert a rigidizing effect.     

Regulation of trout gill AMP deaminase by lipid bilayers. Effects of phospholipid composition and temperature

Trout gill AMP deaminase is inhibited by liposomes made of synthetic phosphatidylcholines containing higher saturated fatty acids. A preincubation of 1 hr, at 4 degrees C, was necessary to obtain the maximal effect. At 4 or 25 degrees C, these phospholipids modified essentially the substrate affinity of the enzyme by increasing the Michaelis constant proportionally to the length of the fatty acid chain. At 13 degrees C, the liposomes decreased the Hill coefficient also, thus inducing a negative cooperativity. Natural phosphatidylcholine and phosphatidylserine were without significant effect on gill AMP deaminase while natural sphingomyelin exhibited a similar effect to that shown in the presence of synthetic phosphatidylcholines. These results are discussed in relation to a possible effect of sphingomyelins in vivo.     

The occurrence of arachidonic acid in Triatoma infestans

1. Gas-liquid chromatography and mass spectrometry were used to verify the presence of arachidonic acid (20:4 omega 6) in adult male and female organs of the blood-suckling bug Triatoma infestans. Fat body, gonad and head lipids were analyzed. 2. Male gonads contained the highest percentage of phospholipids and the highest percentage of arachidonic acid. 3. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingomyelin were isolated from gonads and heads of sexed insects and the fatty acid composition analyzed. 4. All the phospholipids analyzed contained relevant percentages of linoleic acid and fair percentages of arachidonic acid, except for sphingomyelin where no 20:4 omega 6 acid was found. 5. However, arachidonic acid was specifically incorporated in phosphatidylinositol since this phospholipid contained very large percentages of the acid. 6. In male gonads, phosphatidylcholine also contained a similar high percentage of the acid. Arachidonic acid is apparently incorporated, selectively, into these lipids from the blood of the host.     

Acidic phospholipids stimulate the autophosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase

The autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase was stimulated by the acidic phospholipids phosphatidic acid, phosphatidylserine and phosphatidylinositol. Other phospholipids (phosphatidylethanolamine, phosphatidylcholine, sphingomyelin), acidic compounds (dextran sulfate, polyglutamic acid, chondroitin sulfate, hyaluronic acid) and calciumcalmodulin were essentially inactive. Sodium dodecyl sulfate also stimulated the catalytic subunit autophosphorylation, but other detergents (Triton X-100 and deoxycholic acid) did not. The combination of phosphatidic acid and sodium dodecyl sulfate was as effective as each agent alone, suggesting similar stimulation mechanisms. The data suggest that acidic membrane phospholipids might have a role in regulating the autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase.