Plasmodium knowlesi induces alterations in phosphatidylcholine and phosphatidylethanolamine molecular species composition of parasitized monkey erythrocytes

Using high performance liquid chromatography and gas-liquid chromatography, we have characterized the phosphatidylcholine and phosphatidylethanolamine molecular species composition of trophozoite and schizont forms of Plasmodium knowlesi parasitized erythrocytes. Similarly, we determined these parameters in the erythrocyte membranes of trophozoite parasitized cells, unparasitized erythrocytes from infected monkeys before and after a chloroquine treatment and erythrocytes from monkeys that had never been infected. Plasma phosphatidylcholine molecular species composition was also studied. P. knowlesi parasitized erythrocytes presented higher amounts of 16:0/18:2-phosphatidylcholine than the various control cells, which appeared to be compensated for by a decrease in 18:0/20:4-, 16:0/20:3-, 16:0/18:1-, 18:0/18:2-, 18:0/20:3-, 16:0/16:0- and 16:0/18:0-phosphatidylcholines. In the case of phosphatidylethanolamine, the alterations were quantitatively of greater importance and consisted of an increase in, again, 16:0/18:2-phosphatidylethanolamine and a decrease in several species containing 20:4, namely 16:0/20:4-, 18:0/20:4- and 18:1/20:4-phosphatidylethanolamine; also the levels of alkoxy-phosphatidylethanolamines were markedly decreased. P. knowlesi development within monkey erythrocytes therefore appears to be associated with changes in phosphatidylcholine and phosphatidylethanolamine molecular species in the whole parasitized cell. These alterations are also exhibited by the host cell membrane, which provides the first experimental evidence that the parasite is able to manipulate the erythrocyte membrane lipid species composition. The consequences of these alterations on membrane physiology are discussed, as well as the implications that these data may have on the trafficking of phosphatidylcholine and phosphatidylethanolamine in the erythrocytes of P. knowlesi infected monkeys.     

Adaptation of biological membranes to temperature: molecular species compositions of phosphatidylcholine and phosphatidylethanolamine in mitochondrial and microsomal membranes of liver from thermally-acclimated rainbow trout

Summary Molecular species profiles were determined for both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of mitochondrial and microsomal membrane fractions from liver tissue of thermally-acclimated rainbow trout,Salmo gairdneri. The predominant molecular species of PC were 16:0/22:6, 16:0/18:1, 16:0/20:3 and 16:0/22:5, whereas predominant molecular species of PE were 18:1/20:4, 14:0/16:0, 18:0/22:6 and 18:1/22:6. PE possessed short chain saturates (primarily 14:0/16:0) and monoenes (primarily 14:0/16:1) not present in PC and larger proportions of polyunsaturated (18:0/22:6, 18:0/22:5 and 18:1/22:6. and diunsaturated molecular species than PC. Differences between membrane fractions were most evident in warm (20°C)-acclimated trout. Mitochondria contained higher proportions of long-chain, polyunsaturated molecular species of PE, but less of the corresponding species of PC than other membrane fractions. Rankings based on unsaturation index were accordingly: mitochondria heavy microsomes>light microsomes for PE, but heavy microsomes>light microsomes>-mitochondria for PC. Mitochondria were notable for high proportions of diunsaturated molecular species of both phosphatides. Growth at cold temperatures (5°C) was generally associated with a replacement of shorter chain mono- and dienoic molecular species (16:0/18:1, 16:1/18:1, 14:0/16:2 and 18:1/18:1 in the case of PC and 14:0/16:1, 14:0/16:2 and 16:1/18:1 for PE), and occasionally saturates, with long-chain, polyunsaturated molecular species (for PC, C_36–38: 16:0/22:6, 16:1/22:6, 16:0/20:3 and 16:0/20:5; for PE, C_38–40: 18:1/20:4, 16:1/22:6, 18:0/20:5, 18:2/20:4, 18:0/22:5 and 18:0/22:6). However, compositions of mitochondrial PE and PC from heavy microsomes were not significantly influenced by acclimation temperature. The role of phospholipase A₂, in addition to other metabolic processes, in mediating these changes is discussed.Abbreviations ACL average chain length - UI unsaturation index     

Endogenous phospholipids of swine liver: effect of fat deprivation on molecular species of phosphatidylcholine and phosphatidylethanolamine

Three 1-yr-old swine and two 2.5-wk-old swine were fed a fat-free diet for 1 month and 5 months, respectively. The hepatic phosphatidylcholine and phosphatidylethanolamine were fractionated by silver ion thin-layer chromatography. A distinctive feature of the chromatographic procedure was the development of the chromatograms at low temperatures: -10 degrees C for phosphatidylcholine and 4 degrees C for phosphatidylethanolamine. The chromatographic fractions were hydrolyzed with phospholipase A(2), and the fatty acids were characterized. Significant concentrations of odd-chain saturated and unsaturated fatty acids were found in the swine deprived of fat for 5 months. The major molecular species of phosphatidylcholine in both groups contained monoenoic fatty acids: 16:0/18:1(n - 9), 18:0/18:1(n - 9), and 18:1(n - 9)/18:1(n - 9). Their concentrations changed only slightly with the diet. The molecular species of phosphatidylethanolamine were more sensitive to dietary changes. In the swine deprived of fat for 1 month, about 50% of the molecular species of phosphatidylethanolamine contained tetraenoic fatty acids: 16:0/20:4(n - 6), 18:0/20:4(n - 6), and 18:1(n - 9)/20:4(n - 6). The phosphatidylethanolamine of animals deprived of fat for 5 months contained only 3% molecular species with tetraenoic acids, 18:0/20:4(n - 6), but 36% molecular species with trienoic acids: 18:0/20:3(n - 9), 18:1(n - 9)/20:3(n - 9), 18:0/19:3(n - 8), 16:0/20:3(n - 9), and 17:0/20:3(n - 9). Doubly unsaturated species, such as 18:1(n - 9)/18:1(n - 9), 18:1(n - 9)/20:3(n - 9), and 18:1(n - 9)/20:4(n - 6), were found in both groups of swine, although their total concentrations were higher in the group deprived of fat for a longer period.     

Thermal adaptation in biological membranes: interacting effects of temperature and pH

Summary The interacting effects of pH and temperature on membrane fluidity were studied in plasma membranes isolated from liver of rainbow trout (Oncorhynchus mykiss) acclimated to 5 and 20°C. Fluidity was determined as a function of temperature under conditions of both constant (in potassium phosphate buffer) and variable pH (in imidazole buffer, consistent with imidazole alphastat regulation) from the fluorescence anisotropy of two probes: 1,6-diphenyl-1,3,5-hexatriene, which intercalates into the bilayer interior, and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene which is anchored at the membrane/water interface. The temperature dependence of the anisotropy parameter for 1,6-diphenyl-1,3,5-hexatriene in plasma membranes of 20°C-acclimated trout was greater when determined in phosphate (AP per °C=-0.047) than in imidazole buffer (AP per °C=-0.022); similar, but less significant, trends were noted with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene. In contrast, the temperature dependence of fluidity (AP/°C in the range-0.0222 to-0.027) did not vary with buffer composition in membranes of 5°C-acclimated trout. In phosphate buffer, anisotropy parameter values for 1,6-diphenyl-1,3,5-hexatriene were significantly lower in 5°C-than 20°C-acclimated trout, indicating a less restricted probe environment following cold acclimation and nearly perfect compensation (91%) of fluidity. Temperature-dependent patterns of acid-base regulation were estimated to account for 11–40% of the fluidization evident in membranes of 5°C-trout, but a period of cold acclimation was required for complete fluidity compensation. In contrast, no homeoviscous adaptation was evident in imidazole buffer, indicating that membrane fluidity is sensitive to buffer composition. Accordingly, vesicles of bovine brain phosphatidylcholine, suspensions of triolein, and plasma membranes of 5°C-acclimated trout were consistently more fluid in imidazole than phosphate buffer. Membranes of 5°C-acclimated trout were enriched in molecular species of phosphatidylcholine containing 22:6n3 (at the expense of species containing 18:1n9 and 18:2n6) compared to membranes of 20°C-trout; consequently, the unsaturation index was significantly higher (3.29 versus 2.73) in trout maintained at 5 as opposed to 20°C. It is concluded that: 1) the chemical composition of the internal milieu can significantly influence the physical properties of membrane lipids; 2) temperature-dependent patterns of intracellular pH regulation may partially offset the ordering effect of low temperature on membrane fluidity in 20°C-acclimated trout transferred to 5°C, but not in 5°C-acclimated trout transferred to warmer temperatures; 3) the majority of the thermal compensation of plasma membrane fluidity resulting from a period of temperature acclimation most likely reflects differences in membrane composition between acclimation groups; 4) imidazole apparently interacts with trout hepatocyte plasma membranes in a unique way.Abbreviations _im netcharge stateofproteins - AP anisotropyparameter - bw body weight - DPH 1,6-diphenyl-1,3,5-hexatriene - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonicacid - PC phosphatidylcholine - pH_e pHofarterial blood - pH_i intracellular pH - TMA-DPH 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene - TRIS tris(hydroxymethyl)aminomethane     

Fatty acid and phospholipid composition of five psychrotrophic Pseudomonas spp. grown at different temperatures

The free fatty acid and phospholipid composition of 5 psychrotrophic marine Pseudomonas spp. have been determined in chemostat culture with glucose as the limiting substrate over the range 0–20°C. The predominant fatty acid present in all the isolates was hexadecenoic acid (C16:1) together with lesser quantities of octadecenoic acid (C 18:1) whilst none contained acids with chain lengths exceeding 18 carbon atoms. Decreasing the growth temperature from 20°C to 0°C resulted in little significant change in fatty acid composition. The principal phospholipid components of the five psychrotrophic pseudomonads have been identified as phosphatidylserine, phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. Decreasing the growth temperature did not elicit significant changes either in the total quantities of phospholipid synthesized or in the concentration of individual phospholipid components in any of the isolates. All the psychrotrophs showed maximum glucose uptake between 15°C and 20°C and the rate decreased rapidly as the temperature was decreased towards 0°C.Abbreviations PS Phosphatidylserine - PE phosphatidylethanolamine - PG phosphatidylglycerol - DPG diphosphatidylglycerol     

Temperature regulation of oleate desaturase in sunflower (Helianthus annuus L.) seeds

The effect of temperature on oleate desaturation in developing sunflower (Helianthus annuus L.) seeds has been examined. When seeds from plants grown at low (20/10° C, day/night) temperature were transferred for 24 h to 10° C, an increase in the linoleate/oleate ratio in phosphatidylcholine and triacylglycerol was observed, but not when transfer was to 20 or 30° C. The same effect was observed in triacylglycerol, phosphatidylcholine and phosphatidylethanolamine in the newly synthesized lipids after in-vivo incubation with [1-¹⁴C]oleate at 10° C. The microsomal oleoyl phosphatidylcholine desaturase (ODS) activity of the seeds maintained at 10 C was also enhanced. The stimulation was observed after only 3 h in plants grown at high temperature (30/20° C). This effect was inhibited by cycloheximide, implying that the low-temperature stimulation of the ODS activity was caused by the synthesis of new enzyme. As a consequence, seeds from plants grown at low temperature had higher ODS activities and linoleate contents than those grown at high temperature. The microsomal ODS activity of seeds from plants grown at low temperature was dependent on incubation temperature and showed a maximum at 20° C. By contrast, this activity was almost temperature-insensitive in seeds from plants grown at high temperature. These results could explain how temperature regulates the fatty-acid composition in sunflower-seed lipids.Abbreviations DAF days after flowering - ODS oleoyl phosphatidylcholine desaturase - PC phosphatidylcholine - PE phosphatidylethanolamine - TAG triacylglycerol - 181 oleic acid - 182 linoleic acid To whom correspondence should be addressedThanks are due to M.C. Ruiz for skillful technical assistance. This work was supported by a grant from Junta de Andalucia, Spain.     

Isolation and identification of phospholipid molecular species in alpha wild marine shrimp Penaeus kerathurus muscle and cephalothorax

The concentration of TL in Penaeus kerathurus muscle and cephalothorax was 1.03+/-0.04 (75.9+/-0.8% of which was PhL) and 2.36+/-0.07% (45.5+/-0.8% of which was PhL) of the wet tissue, respectively. The phosphatidylethanolamine represented 26.4+/-0.6% (85.6% diacyl- and 14.4% alkyl-acyl- or alkenyl-acyl-analogues) of muscle and 24.7+/-0.2% (90.7% diacyl- and 9.3% alkyl-acyl- or 1-alkenyl-acyl-analogues) of cephalothorax phospholipids while the phosphatidylcholine represented 57.1+/-0.6% (86.9% diacyl- and 13.1% alkyl-acyl- or alkenyl-acyl-analogues) of muscle and 47.2+/-0.4% (89.1% diacyl- and 10.9% alkyl-acyl- or 1-alkenyl-acyl-analogues) of cephalothorax phospholipids, respectively. The main fatty acids of phosphatidylethanolamine were C16:0, C18:0, C18:1 omega-9, C20:4 omega-6, C20:5 omega-3, C22:6 omega-3 and of phosphatidylcholine were C16:0, C18:0, C18:1 omega-9, C20:4 omega-6, C20:5 omega-3. Low percentages of 2-OH C14:0 and cyclo-17:0 fatty acids were also determined. Phosphatidylethanolamine were found to contain a significantly (P<0.05) higher percentage of polyunsaturated fatty acids compared to phosphatidylcholine. The omega-3/omega-6 ratio in muscle phosphatidylethanolamine and phosphatidylcholine was significantly (P<0.05) higher to the ones of cephalothorax.     

Composition and incorporation of [3H]arachidonic acid into molecular species of phospholipid classes by cultured human endothelial cells

Based on quantitative high-performance liquid chromatographic analyses of molecular species in selected phospholipid subclasses from culture human umbilical vein endothelial cells, the relative degree of unsaturation was ethanolamine plasmalogens greater than phosphatidylethanolamine greater than phosphatidylcholine. A total of 36 different molecular species were identified in the phosphatidylcholine fraction. Interestingly, the phosphatidylcholine contained a significant amount (11.7%) of the dipalmitoyl species, a lipid normally associated with lung surfactant. The arachidonoyl-containing molecular species of phosphatidylserine/inositol were labeled to the highest extent and the ethanolamine plasmalogens contained the lowest specific radioactivity after incubating [3H]arachidonic acid with human endothelial cells for 4 h. Within each phospholipid subclass the arachidonoyl species where both acyl groups of the phospholipid are unsaturated (20:4-20:4, 18:2-20:4 + 16:1-20:4, and 18:1-20:4) had higher specific radioactivities, after labeling with [3H]arachidonic acid, than those that contained saturated aliphatic chains (16:0-20:4 and 18:0-20:4). This indicates that the unsaturated species have higher turnover rates.     

Composition of phospholipid molecular species from mammary tumors

The molecular phospholipid species of mammary tumors induced by 7,12-dimethylbenz[a]anthracene in rats that were fed diets containing 20 or 3% sunflower-seed oil and different levels of calcium were analyzed by high-pressure liquid chromatography. Twenty-seven molecular species of phospholipids were identified. Phosphatidylcholine was predominantly composed of palmitoyl-arachidonoyl (16:0-20:4) (17-21%), palmitoyl-oleoyl (16:0-18:1) (19-21%), stearoyl-arachidonoyl (18:0-20:4) (12-13%), and 1,2-dipalmitoyl (16:0-16:0) (10-14%) species. The major molecular species of phosphatidylethanolamine were 18:0-20:4 (37-39%) and 16:0-20:4 (10-11%). The composition of diacyl phosphatidylcholine and diacyl phosphatidylethanolamine molecular species from rat mammary tumors was not greatly affected by the different diets.     

Characteristics of phospholipids in microvillar membranes of octopus photoreceptor cells.

Characteristics of lipids in the microvillar membranes of octopus photoreceptor cells were studied in order to obtain some information on the membrane environment with rhodopsin in the invertebrate. (1) The membranes contain lipid and protein in almost equal proportion. The majority of lipids are phospholipids. Neutral lipids make up 16% of the total lipids, the major constituent of which is cholesterol. (2) Phosphatidylethanolamine and phosphatidylcholine are the major phospholipids. Phosphatidylserine, ceramide 2-aminoethylphosphonate and sphingomyelin occur as minor components. An unidentified alkaline and acid stable phospholipid was found. (3) The predominant fatty acids of phosphatidylethanolamine and phosphatidylcholine are highly unsaturated such as 22 : 6, 20 : 5 and 20 : 4. The 22 : 6 and 20 : 5 are exclusively linked at the 2-position, but the 20 : 4 is linked significantly at the 1-position of the phospholipids. (4) Major molecular species are 16 : 0/22 : 6 (48.4%) and 16 : 0/20 : 4 (19.6%) in phosphatidylcholine, and 20 : 4/22 : 6 (50.7%) and 16 : 0/22 : 6 (25.6%) in phosphatidylethanolamine.     

The effects of temperature on the fatty acid and phospholipid composition of four obligately psychrophilicVibrio Spp.

The free fatty acid and phospholipid composition of 4 psychrophilic marineVibrio spp. have been determined in chemostat culture with glucose as the limiting substrate over a temperature range 0–20°C. All the isolates show maximum glucose and lactose uptake at 0°C and this correlates with maximum cell yield. None of the isolates contain fatty acids with a chain length exceeding 17 carbon atoms.Vibrio AF-1 andVibrio AM-1 respond to decreased growth temperatures by synthesizing increased proportions of unsaturated fatty acids (C15:1, C16:1 and C17:1) whereas inVibrio BM-2 the fatty acids undergo chain length shortening. The fourth isolate (Vibrio BM-4) contains high levels (60%) of hexadecenoic acid at all growth temperatures and the fatty acid composition changes little with decreasing temperature. The principal phospholipid components of the four psychrophilic vibrios were phosphatidylserine, phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. Lyso-phosphatidylethanolamine and 2 unknown phospholipids were additionally found inVibrio AF-1. The most profound effect of temperature on the phospholipid composition of these organisms was the marked increase in the total quantities synthesized at 0°C. At 15°C phosphatidylglycerol accumulated in the isolates as diphosphatidylglycerol levels decreased. Additionally inVibrio BM-2 andVibro BM-4 phosphatidylserine accumulates as phosphatidylethanolamine biosynthesis was similarly impaired. The observed changes in fatty acid and phospholipid composition in these organisms at 0°C may explain how solute transport is maintained at low temperature.Abbreviations PS Phosphatidylserine - PE phosphatidylethanolamine - PG phosphatidylglycerol - DPG diphosphatidylglycerol - lyso PE lysophosphatidylethanolamine     

RADIOLABELING STUDIES OF LIPIDS AND FATTY ACIDS IN NANNOCHLOROPSIS (EUSTIGMATOPHYCEAE), AN OLEAGINOUS MARINE ALGA1

The synthesis of fatty acids and lipids in Nannochloropsis sp. was investigated by labeling cells in vivo with [¹⁴C]-bicarbonate or [¹⁴C]-acetate. [¹⁴C]-bicarbonate was incorporated to the greatest extent into 16:0, 16:1, and 14:0 fatty acids, which are the predominant fatty acids of triacylglycerols. However, more than half of the [¹⁴C]-acetate was incorporated into longer and more desaturated fatty acids, which are constituents of membrane lipids. [¹⁴C]-acetate was incorporated most strongly into phosphatidylcholine, which rapidly lost label during a 5-h chase period. The label associated with phosphatidylethanolamine also decreased during the chase period, whereas label in other membrane lipids and triacylglycerol increased. The dynamics of labeling, along with information regarding the acyl compositions of various lipids, suggests that 1) the primary products of chloroplast fatty acid synthesis are 14:0, 16:0, and 16:1; 2) C₂₀ fatty acids are formed by an elongation reaction that can utilize externally supplied acetate; 3) phosphatidylcholine is a site for desaturation of C₁₈ fatty acids; and 4) phosphatidylethanolamine may be a site for desaturation of C₂₀ fatty acids.     

Properties of microsomal acyl-CoA: lysophosphatidylcholine acyltransferase from liver of thermally-acclimated rainbow trout,Salmo gairdneri

Summary Acyl-CoA: lysophosphatidylcholine acyltransferase (LPCAT) (EC 2.3.1.23) activity was assayed in liver microsomes from rainbow trout,Salmo gairdneri, acclimated to 5°C and 20°C to assess its contribution to the temperature-induced restructuring of phospholipid acyl chain composition. The synthesis of phosphatidylcholine (PC) (from lyso-PC) was threefold the synthesis of phosphatidylethanolamine (PE) (from lyso-PE) under similar assay conditions. LPCAT activity (i) displayed an absolute requirement for lysophosphatidylcholine (LPC) and was enhanced by the presence of ATP, MgCl₂ and CoA (which reduced the impact of endogenous acyl-CoA hydrolase activity by regenerating the acyl-CoA substrate) in the assay medium; (ii) remained linear with time up to 30 min; and (iii) increased linearly with microsomal protein concentration up to 0.2 mg/ml for the 20°C assay and 0.4 mg/ml for the 5°C assay. There was no difference in K_m or V_max values due to the acclimation history of the fish, but there were obvious differences due to assay temperature. The apparent K_m values for LPC were 58.54±7.24 M and 12.26±2.14 M when assayed at 5°C and 20°C respectively; values for oleoyl-CoA were 9.11±0.78 M and 1.23±0.25 M under the same assay conditions. Activity was 1.99±0.31 nmol min^–1 mg protein^–1 when assayed at 5°C, and 3.8±0.45 nmol min^–1 mg protein^–1 when assayed at 20°C. These findings indicate that adjustments in the activity of LPCAT play no significant role in the temperature-induced restructuring of PC molecular species composition. However, the marked temperature dependence of the K_m values for LPC and oleoyl CoA suggest that patterns of fatty acid incorporation (i.e. substrate preference) may vary with assay temperature, and in this way LPCAT could contribute to the restructuring response.Abbreviations PC phosphatidylcholine - PE phosphatidylethanolamine - LPCAT acyl-CoA: lysophosphatidylcholine acyltransferase - LPEAT acyl-CoA: lysophosphatidylethanolamine acyltransferase - LPC 1-palmitoyl,2-lysophosphatidylcholine     

Molecular Species of Diacylglycerols and Phosphoglycerides and the Postmortem Changes in the Molecular Species of Diacylglycerols in Rat Brains

The molecular species of 1,2-diacyl-sn-glycerol (DAG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) from brains of adult rats (weighing 150 g) were determined. The DAG, isolated from brain lipid extracts by TLC, was benzoylated, and the molecular species of the purified benzoylated derivatives were separated from each other by reverse-phase HPLC. The total amount and the concentration of each species were quantified by using 1,2-distearoyl-sn-glycerol (18:0-18:0) as an internal standard. About 30 different molecular species containing different fatty acids at the sn-1 and sn-2 positions of DAG were identified in rat brains (1 min postmortem), and the predominant ones were 18:0-20:4 (35%), 16:0-18:1 (15%), 16:0-16:0 (9%), and 16:0-20:4 (8%). The molecular species of PC, PE, PS, and PI were determined by hydrolyzing the lipids with phospholipase C to DAG, which was then benzoylated and subjected to reverse-phase HPLC. PIP and PIP2 were first dephosphorylated to PI with alkaline phosphatase before hydrolysis by phospholipase C. The molecular species composition of phosphoinositides showed predominantly the 18:0-20:4 species (50% in PI and approximately 65% in PIP and PIP2). PS contained mainly the 18:0-22:6 (42%) and 18:0-18:1 (24%) species. PE was mainly composed of the 18:0-20:4 (22%), 18:0-22:6 (18%), 16:0-18:1 (15%), and 18:0-18:1 (15%) species. In PC the main molecular species were 16:0-18:1 (36%), 16:0-16:0 (19%), and 18:0-18:1 (14%). Studies on postmortem brains (30 s to 30 min) showed a rapid increase in the total amount (from 40-50 nmol/g in 0 min to 210-290 nmol/g in 30 min) and in all the molecular species of DAG. Comparatively larger increases (seven- to 10-fold) were found for the 18:0-20:4 and 16:0-20:4 species. Comparison of DAG species with the molecular species of different glycerolipids indicated that the rapid postmortem increase in content of DAG was mainly due to the breakdown of phosphoinositides. However, a slow but continuous breakdown of PC to DAG was also observed.     

Hormonal stimulation of diacylglycerol formation in hepatocytes. Evidence for phosphatidylcholine breakdown

The molecular species of 1,2-diacylglycerol in control and agonist-stimulated rat hepatocytes were analyzed by high performance liquid chromatography. Twelve species were identified which were increased nonuniformly by 100 nM vasopressin. Most species were increased 2-3-fold, but some (C16:0/C20:4 and C18:0/C20:4) were increased 3-6-fold. Selectively greater increases in the latter two species were also induced by ATP, angiotensin II, and A23187 ionophore, however, phorbol ester caused uniform increases. Calcium depletion of the cells with chelator resulted in a uniform 2-fold effect of vasopressin on 1,2-diacylglycerol species, with greater increases in C16:0/C20:4 and C18:0/C20:4 being restored by Ca2+ readdition. Comparison of the increases in 1,2-diacylglycerol species caused by the Ca2+-mediated agents with the molecular species present in rat hepatocyte phospholipids supports the concept that phosphatidylcholine is a major source of the 1,2-diacylglycerol that accumulates. In hepatocytes incubated for 5 min to 2 h with 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine, the label was incorporated mainly into phosphatidylcholine, and subsequent incubation with vasopressin, angiotensin II, ATP, epinephrine, A23187, and phorbol ester caused formation of [3H]alkyl-acylglycerol, but not [3H]alkyl-phosphatidic acid. The time course and concentration dependence of the vasopressin effect were similar to those reported previously for total 1,2-diacylglycerol (Bocckino, S. B., Blackmore, P. F., and Exton, J. H. (1985) J. Biol. Chem. 260, 14201-14207). Calcium depletion induced by chelator inhibited the effect of vasopressin, and readdition of Ca2+ largely restored the effect. In cells incubated with [14C]lyso-phosphatidylcholine, [3H]phosphatidylcholine, or [14C]phosphatidylethanolamine for 5 or 30 min to label hepatocyte phosphatidylcholine, vasopressin also induced the formation of labeled 1,2-diacylglycerol, but not phosphatidic acid. In contrast, in hepatocytes prepared from rats injected intraportally with [3H]alkyl-lyso-glycerophosphocholine 20 h previously, the hormone induced the rapid formation of both labeled 1,2-diacylglycerol and phosphatidic acid. In summary, these isotopic data indicate that a rapidly labeled pool of phosphatidylcholine is hydrolyzed to 1,2-diacylglycerol and a slowly labeled pool is broken down to both 1,2-diacylglycerol and phosphatidic acid in hepatocytes stimulated by Ca2+-mobilizing agents. It is concluded from both the analyses of molecular species of 1,2-diacylglycerol and the labeling experiments that phosphatidylcholine is a major source of the 1,2-diacylglycerol that accumulates in hepatocytes stimulated with Ca2+-mobilizing agonists and that the mechanisms responsible may involve both Ca2+ and protein kinase C.     

Selective changes to phosphatidylcholine and phosphatidylethanolamine molecular species in the developing fetal guinea pig liver and plasma

The molecular species composition of membrane phospholipids influences the activities of integral proteins and cell signalling pathways. We determined the effect of increasing gestational age on fetal guinea pig liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and plasma PC molecular species composition. The livers were collected from fetuses (n = 5/time point) at 5 day intervals between 40 and 65 days of gestation, and at term (68 days). Hepatic PC and PE molecular species composition was determined by electrospray ionisation mass spectrometry. An increasing gestational age was accompanied by selective changes in individual molecular species. The proportion of the sn-1 18:0 species increased relative to the sn-1 16:0 species in liver PC, but not PE, with an increasing gestational age. 1-O-alkyl-2-acyl PC species concentrations decreased significantly between 40 and 45 days of gestation (40%), and 65 and 68 days (54%). Total 1-O-alkenyl-2-acyl PE species concentration increased between days 60 and 65, due to a rise in 1-O-16:0 alkyl/20:4 content, and then decreased until term. Between day 40 and term, PC and PE sn-2 18:2n-6 species concentrations increased 3-fold. PC16:0/18:2 increased gradually throughout gestation, while PC18:0/18:2 content only increased after day 65. The overall increase in PE18:2n-6 content was due to PE18:0/18:2 alone. The composition of plasma PC essentially reflected hepatic PC. Overall, these data suggest differential regulation of hepatic PC and PE molecular species composition during development which is essentially independent of the maternal fatty acid supply.     

Endonuclear lipids in liver cells

Lipids are not only components of cell nucleus membranes, but are also found in the membrane-depleted nuclei where they fulfill special functions. We have investigated the lipid composition of membrane-depleted rat liver nuclei obtained by incubation with low Triton X-100 concentrations of 0.04% and 0.08%, which rendered them unaltered or hardly altered. Under these conditions, 26% of proteins and 22% of phospholipids were recovered. The main phospholipids were phosphatidylcholine > phosphatidylethanolamine > phosphatidylinositol = or > phosphatidylserine and sphingomyelin (in decreasing concentrations). The fatty acid components of total lipids and phosphatidylcholine were mainly unsaturated. Over 40% belonged to the n-6 series (arachidonic > or = 25% and linoleic 15%); approximately 40% corresponded to saturated acids and <10% were monoenoic. Endonuclear phosphatidylcholine was built up by 16 molecular species, the most abundant being 18:0-20:4 (32%), 16:0-20:4 (19%), 16:0-18:2 (13%), and 18:0-18:2 (11%). The fatty acid composition and phosphatidylcholine molecular species distribution in the membrane-depleted nucleus of rat liver showed patterns similar to the whole nucleus, mitochondria, microsomes, and homogenate of the parent liver cells, suggesting that endonuclear lipid pool composition is mainly determined by a liver organ profile.     

Evidence that only newly made phosphatidylethanolamine is methylated to phosphatidylcholine and that phosphatidylethanolamine is not significantly deacylated-reacylated in rat hepatocytes

The metabolism of the molecular species of phosphatidylethanolamine derived from [3H]ethanolamine and molecular species of phosphatidylcholine derived from [3H]ethanolamine or [methyl-3H]choline has been studied in rat hepatocytes. After an initial pulse of radioactivity for 1 h and a chase for up to 24 h, the cells were harvested and the incorporation of label into the various molecular species of phosphatidylethanolamine and phosphatidylcholine was determined. The incorporation and metabolism of choline- and ethanolamine-labeled phosphatidylcholine was consistent with deacylation of some species of phosphatidylcholine and reacylation to form molecular species of phosphatidylcholine with different fatty acyl components. In contrast, such remodeling of ethanolamine-labeled phosphatidylethanolamine was not evident. Radioactivity disappeared from all molecular species of phosphatidylethanolamine without an increase in any of the species of phosphatidylethanolamine. This radioactivity was recovered in water-soluble metabolites in the cells and medium. Phosphatidylethanolamine (16:0-22:6) had an initial turnover rate (5.8 nmol/h) which was two or more times that of any of the other major molecular species of phosphatidylethanolamine. The molecular species of phosphatidylethanolamine displayed biphasic turnover profiles. The second rate of decay of radioactivity between 12 and 24 h was 2-4 times slower than the initial decay rate. During the first 2 h of the chase period, phosphatidylcholine was a major metabolite of labeled phosphatidylethanolamine. Subsequently, there was minimal conversion of phosphatidylethanolamine to phosphatidylcholine which suggests that only newly made phosphatidylethanolamine is available as a substrate for methylation to phosphatidylcholine.     

Membrane phospholipid asymmetry in Semliki Forest virus grown in BHK cells

The distribution of phospholipids across the membrane bilayer of Semliki Forest virus grown in BHK cells has been examined by treating the virus with bee venom phospholipase A₂ and sphingomyelinase C from Staphylococcus aureus. From the amounts of different phospholipids which are degraded rapidly (half-time about 1 min for phospholipase A₂) we calculate that in virus isolated 16 h after infection about 95% of sphingomyelin, 55% of phosphatidylcholine, 20% of phosphatidylethanolamine and less then 5% of phosphatidylserine is present on the outer leaflet of the virus envelope. Less than 5% of the virus was permeable to macromolecules before or after treatment with phospholipases as judged by accessibility of the genome to external ribonuclease. A much slower (half-time about 1 h) breakdown by phospholipase A₂ of originally inaccessible phosphatidylcholine and phosphatidylethanolamine appeared to be due to an enzyme-induced loss of lipid asymmetry since the original asymmetric distribution of phospholipids was maintained for several hours when the virus alone was incubated at 37°C. However, virus incubated for 20 h at 37°C showed a marked loss of phosphatidylethanolamine and phosphatidylserine asymmetry and a greater susceptibility to lysis by longer treatment with phospholipase A₂.