Metabolism of unique diarachidonoyl and linoleoylarachidonoyl species of ethanolamine and choline phosphoglycerides in rat testes

Selected molecular species of rat testicular 1,2-diradyl-sn-glycero-3-phosphocholines and 1,2-diradyl-sn-glycero-3-phosphoethanolamines were quantitated as their diradylglycerobenzoate derivatives, using a recently developed high-performance liquid chromatographic method. Increased amounts of docosapentaenoic acid were found in glycerophospholipids containing ether moieties compared with the diacyl phospholipids (e.g., docosapentaenoate-containing species comprised more than 80% of the alkylacyl subclass of the ethanolamine phosholipids as opposed to 29.3% of the diacyl subclass). Within 2 h after intratesticular injections of [5,6,8,9,11,12,14,15-3H]arachidonic acid, the 20:4-20:4 and 18:2-20:4 molecular species of the diacyl subclass of both the choline and ethanolamine glycerophosphatides had the highest specific radioactivities. These unique molecular species (20:4-20:4 and 18:2-20:4) also exhibited the largest percentage decrease in specific radioactivity 24 h after the intratesticular injections of [3H]arachidonic acid, which indicates these two species possess a high metabolic turnover. Two of the arachidonate-containing molecular species (18:1-20:4 and 18:0-20:4) in the ethanolamine plasmalogens showed only a small decrease in specific radioactivity, whereas a third species (16:0-20:4) actually had a 44% increase in specific radioactivity 24 h after the intratesticular injections of [3H]arachidonate. These data indicate that the 20:4-20:4, 18:2-20:4 and 18:1-20:4 species of phosphatidylcholine and/or phosphatidylethanolamine are most rapidly labeled after administration of [3H]arachidonic acid and that they appear to serve as the source of the [3H]arachidonate that is ultimately transferred to ethanolamine plasmalogens.     

Highly unsaturated phospholipid molecular species of rat erythrocyte membranes: selective incorporation of arachidonic acid into phosphoglycerides containing polyunsaturation in both acyl chains

This study describes for the first time the complete molecular species composition and turnover of [3H]arachidonic acid in various glycerophospholipid classes of rat erythrocytes, a model system that has been extensively used to investigate numerous membrane phenomena. Quantitative analysis of the individual molecular species of the choline, ethanolamine, serine, and inositol glycerophospholipid classes was possible by preparing their diradylglycerobenzoate derivatives that can be quantitated by on-line uv detection in conjunction with high-performance liquid chromatography; turnover of the molecular species containing arachidonate was evaluated in erythrocytes labeled with [3H]arachidonic acid. A unique observation was the significant amounts of 22:6-20:4, 20:4-20:4, and 18:2-20:4 species observed in the diacyl fractions of phosphatidylethanolamine and phosphatidylserine. Moreover, the analysis of the specific radioactivities of individual phospholipid species from erythrocytes incubated with [3H]arachidonic acid demonstrated a selective incorporation of arachidonic acid into the most highly unsaturated molecular species in all of the phospholipid classes examined. Although the 22:6-20:4, 20:4-20:4, and 18:2-20:4 species represented only 4.5% of the total mass of the diacyl phosphoglycerides, these species accounted for a major portion (37%) of the arachidonic acid incorporated into the phospholipids. These results demonstrate the existence of unique populations of phospholipid molecules in rat erythrocytes with a high degree of unsaturation that exhibit a very rapid metabolic turnover rate.     

Heterogeneity in the metabolism of the arachidonoyl molecular species of glycerophospholipids of rabbit alveolar macrophages. The interrelationship between metabolic activities and chemical structures of the arachidonoyl molecular species

The relative incorporation of [3H]arachidonic acid (20:4) into individual molecular species containing 20:4 at the 2 position (18:1-20:4, 16:0-20:4 and 18:0-20:4 species) of diacyl and ether-linked glycerophosphocholine, glycerophosphoethanolamine and glycerophosphoinositol of rabbit alveolar macrophages has been measured by reversed-phase high-performance liquid chromatography (HPLC). The rate of incorporation of [3H]20:4 into the molecular species of glycerophospholipids was greatly influenced by their structures. The reversed-phase HPLC analysis allowed elucidation of the influence of structural differences, such as the nature of the polar head group, the fatty chain at the 1 position and the chemical form of the bond of the fatty chain attached at the 1 position on the uptake of [3H]20:4 by comparison of the specific radioactivities of arachidonoyl molecular species having the same structures, except that one of the three kinds of moiety was different. The specific radioactivities of the molecular species containing choline head groups were significantly higher than those containing ethanolamine and inositol moieties. The specific radioactivities of diacyl molecular species were considerably higher than those of ether-linked molecular species. The nature of the fatty chain attached at the 1 position also influenced the uptake of [3H]20:4 into glycerophospholipids. The arachidonoyl molecular species containing 18:1 at the 1 position were preferentially labelled with [3H]20:4 as compared to the corresponding 16:0-20:4 and 18:0-20:4 species either of diacyl or ether-linked glycerophospholipids. The present results suggest that the acyltransferase involved in the incorporation of 20:4 into glycerophospholipids has selectivity for the structures of glycerophospholipids and the order of selectivity of this enzyme for the arachidonoyl molecular species, deduced in the present experiments, was as follows: choline head group greater than ethanolamine and inositol groups, acyl bond greater than ether and vinyl ether bonds, 18:1 fatty chain greater than 16:0 and 18:0 fatty chains at the 1 position. Comparison of the metabolic activities of all major arachidonoyl molecular species of glycerophospholipids having a single structure is reported here for the first time.     

Identification of Two Molecular Species of Rat Brain Phosphatidylcholine that Rapidly Incorporate and Turn Over Arachidonic Acid In Vivo

Abstract: In vivo rates of arachidonic acid incorporation and turnover were determined for molecular species of rat brain phosphatidylcholine (PtdCho) and phosphatidylinositol (PtdIns). [³H]Arachidonic acid was infused intravenously in pentobarbital-anesthetized rats at a programmed rate to maintain constant plasma specific activity for 2–10 min. At the end of infusion, animals were killed by microwave irradiation, and brain phospholipids were isolated, converted to diacylglycerobenzoates, and resolved as molecular species by reversed-phase HPLC. Most [³H]arachidonate (>87%) was incorporated into PtdCho and PtdIns, with arachidonic acid at the sn -2 position and with oleic acid (18:1), palmitic acid (16:0), or stearic acid (18:0) at the sn -1 position. However, 10–15% of labeled brain PtdCho eluted in a small peak containing two molecular species with arachidonic acid at the sn -2 position and palmitoleic acid (16:1) or linoleic acid (18:2) at the sn -1 position. Analysis demonstrated that tracer was present in both the 16:1–20:4 and 18:2–20:4 PtdCho species at specific activities 10–40 times that of the other phospholipids. Based on the measured mass of arachidonate in each phospholipid molecular species, half-lives were calculated for arachidonate of <10 min in 16:1–20:4 and 18:2–20:4 PtdCho and 1–3 h in 16:0–20:4, 18:0–20:4, and 18:1–20:4 PtdCho and PtdIns. The very short half-lives for arachidonate in the 16:1–20:4 and 18:2–20:4 PtdCho molecular species suggest important roles for these molecules in brain phospholipid metabolism and signal transduction.     

Differential turnover of polyunsaturated fatty acids in plasmalogen and diacyl glycerophospholipids of isolated cardiac myocytes

To investigate the relative turnover of esterified polyunsaturated fatty acids in diacylglycerophospholipids and plasmalogens in isolated cardiac myocytes, we characterized the phospholipid composition and distribution of radiolabel in different phospholipid classes and in individual molecular species of diradyl choline (CGP) and ethanolamine (EGP) glycerophospholipids after incubation of isolated cardiac myocytes with [3H]arachidonate or [14C]linoleate. Plasmalogens in CGP (55%) and EGP (42%) quantitatively accounted for the total plasmalogen content (39%) of cardiac myocyte phospholipids. Plasmalogens comprised 86% and 51% of total arachidonylated CGP and EGP mass, respectively, and [3H]arachidonate was primarily incorporated into plasmalogens in both CGP (65%) and EGP (61%) classes. The specificity activity of [3H]arachidonylated diacyl-CGP was approximately 2- to 5-fold greater than that of [3H]arachidonylated choline plasmalogen, whereas comparable specific activities were found in the [3H]arachidonate-labeled ethanolamine plasmalogen and diacyl-EGP pools. Of the total linoleate-containing CGP and EGP mass, 54% and 57%, respectively, was esterified to plasmalogen molecular species. However, [14C]linoleate was almost exclusively incorporated into diacyl-CGP (96%) and diacyl-EGP (86%). The specific activities of [14C]linoleate-labeled diacyl-CGP and diacyl-EGP were 5- to 20-fold greater than that of the [14C]linoleate-labeled plasmalogen pools. The differential incorporation of polyunsaturated fatty acids in plasmalogens and diacylglycerophospholipids demonstrates that the metabolism of the sn-2 fatty acyl moiety in these phospholipid subclasses is differentially regulated, possibly fulfilling separate and distinct physiologic roles.     

Different metabolic rates for arachidonoyl molecular species of ethanolamine glycerophospholipids in rat brain

The ethanolamine glycerophospholipids (EGP) contain most of the arachidonate (20:4, n-6) and adrenate (22:4, n-6), potential precursors of biologically potent prostaglandins and related compounds. Much better methods utilizing high performance liquid chromatography (HPLC) techniques are now available for the study of the molecular species of all three classes, namely diacyl, alkenylacyl (plasmalogen), and alkylacyl. Different molecular species may have different functions. This possibility was studied by examining the rates of incorporation of [3H]arachidonic acid into the three major molecular species of each of the three classes of ethanolamine glycerophospholipids. After the intracerebral injection of [3H]20:4 into rat brain, it was rapidly converted to 22:4(n-6). Of the total radioactivity, 10-20% was located in 22:4 in alkenylacyl and diacyl-GPE. In the alkylacyl-GPE, labeled 22:4 was preferentially incorporated and accounted for 50-60% of the total radioactivity. The primary arachidonoyl molecular species of alkenylacyl, alkylacyl, and diacyl-GPE were the 18:1-20:4, 16:0-20:4, and 18:0-20:4 species. The alkylacyl class contained almost equal proportions of these three molecular species. On the other hand, the 20:4 in alkenylacyl and diacyl classes was combined largely with 18:0 groups at the sn-1 position. In particular, the 18:0-20:4 species comprised about 80% of arachidonoyl molecular species of the diacyl class. In all three classes, the highest specific radioactivities were found in the 18:1-20:4 species, whereas the 18:0-20:4 species had the lowest specific radioactivity. Over the period 60 min-24 hr, the diacyl 18:0-20:4 and all three arachidonoyl molecular species of the alkenylacyl class increased in specific radioactivity more rapidly than the other arachidonoyl molecular species.     

Bradykinin-stimulated release of [3H]arachidonic acid from phospholipids of HSDM1C1 cells: comparison of diacyl phospholipids and plasmalogens as sources of prostaglandin precursors

Ethanolamine plasmalogens (1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamines) of many tissues contain high levels of arachidonate at their 2-position, and in certain tissues have been implicated as possible donors of arachidonate required in the synthesis of prostaglandins and thromboxanes. In the present study, [3H]arachidonate-labeled phospholipids of HSDM1C1 cells, a cell line derived from a mouse fibrosarcoma, were examined to determine the donor of the arachidonic acid released upon bradykinin stimulation of the synthesis of PGE2. HSDM1C1 cells labeled with [3H]arachidonic acid for 24 hr in serum-free medium were used in most of the experiments and had the following distribution of label among the cellular lipids; phosphatidylcholine (33%), phosphatidylinositol (20%), diacyl-sn-glycero-3-phosphoethanolamine (15%), ethanolamine plasmalogen (15%), and less polar lipids )16%). Bradykinin treatment stimulated a rapid hydrolysis of [3H]arachidonate from the cellular lipids and conversion of the released acid to PGE2, which was secreted into the medium. The label was released predominantly from phosphatidylinositol and possibly from phosphatidylcholine with no detectable change in the labeling of diacyl- or 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. The ethanolamine plasmalogens, therefore, do not appear to be involved in the stimulated release of arachidonate in the HSDM1C1 cells. Indomethacin blocked the bradykinin-stimulated synthesis of PGE2 and to a lesser degree inhibited the release of [3H]arachidonate from the cellular lipids into the medium.     

Serine and ethanolamine incorporation into different plasmalogen pools: Subcellular analyses of phosphoglyceride synthesis in cultured glioma cells

In cultured glioma cells, plasma membrane (PM) is enriched in phosphatidylserine (PtdSer) and plasmalogens (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine). Serine can be a precursor of headgroups of both ptdSer and ethanolamine phosphoglycerides (PE) including plasmalogens and non-plasmalogen PE (NP-PE). Synthesis of phospholipids was investigated at the subcellular level using established fractionation procedures and incorporation of [³H(G)]L-serine and [1,2-¹⁴C]ethanolamine. Specific radioactivity of PtdSer from [³H]serine was 2-fold greater in PM than in microsomes, reaching maximum by 2–4 h. Labeled plasmalogen from [³H]serine appeared in PM by 4 h and increased to 48 h, whereas almost no plasmalogen accumulated in microsomes within 12 h. In contrast, labeled plasmalogen from [1,2-¹⁴C]ethanolamine appeared in both PM and microsomes at early incubation times and became enriched in PM beyond 12 h. Thus, in glioma cells: (1) greater and faster accumulation of labeled PtdSer in PM may reflect direct synthesis from serine within PM; (2) PM is a major source of PtdSer for decarboxylation and PE synthesis; (3) NP-PE in both PM and microsome provides headgroup for synthesis of plasmalogen; and, (4) plasmalogen synthesis may involve different intracellular pools depending on headgroup origin.Abbreviations NP-PE nonplasmenylethanolamine phosphoglycerides including both diacyl and alkylacyl species - PE total ethanolamine phosphoglycerides: plasmalogen-plasmenylethanolamine or alkenylacyl ethanolamine phosphoglyceride (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) - PL phospholipid - PM plasma membrane - PtdCho phosphatidylcholine - PtdSer phosphatidylserine     

Studies on the enzymatic pathways of calcium ionophore-induced phospholipid degradation and arachidonic acid mobilization in peritoneal macrophages

Exposure of mouse peritoneal macrophages to ionophore A23187 caused a rapid and extensive Ca2+-dependent phospholipid degradation and mobilization of arachidonic acid. Phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine all contributed to the arachidonic acid release, although the ethanolamine phospholipids incorporated [3H]arachidonic acid more slowly during the prelabeling period, particularly the plasmalogen form. Several enzymatic pathways could be positively identified as contributing to the ionophore-induced phospholipid degradation by the use of several different radiolabeled phospholipid precursors: (i) a phospholipase A-mediated deacylation, (ii) a phosphodiesterase (phospholipase C) reaction, rapidly generating diacylglycerol units from inositol phospholipids, and (iii) enzymatic processes generating diacylglycerol and CDP- and phosphocholine/ethanolamine from phosphatidylcholine/ethanolamine. The diacylglycerol formed was in part phosphorylated and in part hydrolyzed to monoacylglycerol, with retention of its arachidonic acid. These, and other, results indicate that the Ca2+-ionophore activates several apparently distinct phospholipid-degrading processes, in contrast to stimuli acting via cellular receptors.     

Effect of 1,25-dihydroxyvitamin D3 on phospholipid metabolism in chick duodenal mucosal cell. Relationship to its mechanism of action

Pretreatment of the D-deficient chick with 1,25-dihydroxyvitamin D3 increases de novo synthesis of phosphatidylcholine by a stimulation of CDP-choline: sn-1,2-diacylglycerol choline-phosphotransferase reaction. The time course of change in the incorporation of [3H]choline and [14C]ethanolamine into the brush border lipid fraction after 1,25-dihydroxyvitamin D3 treatment correlates closely with the time course of change in calcium uptake into the brush border membrane vesicles. Prior treatment with cycloheximide does not block this increase in phosphatidylcholine synthesis. In addition, 1,25-dihydroxyvitamin D3 administration increases the incorporation of [3H]arachidonic acid into the phosphatidylcholine fraction of the brush border to a great extent but does not increase the incorporation of [3H]palmitic acid into the phosphatidylcholine fraction. The incorporation of these 3H labeled fatty acids into diacylglycerol is not changed by 1,25-dihydroxyvitamin D3. These data indicate that 1,25-dihydroxyvitamin D3 enhances the synthesis of phosphatidylcholine independent of new protein synthesis, and also increases the incorporation of unsaturated fatty acids into phosphatidylcholine. From these results we suggest that changes in phospholipid metabolism in the enterocyte are the mechanisms by which 1,25-dihydroxyvitamin D3 acts to enhance calcium entry across the brush border membrane.     

Phosphatidyl choline fatty acid remodeling in the hepatic cell nuclei

This study was performed to determine whether fatty acids incorporated into liver cell nuclei phosphatidylcholine (PtdCho) could be remodeled in the isolated nuclear. For this reason, rat liver cell nuclei were incubated in vitro with [1-14C]20:4n-6-CoA. PtdCho molecular species with the highest specific activity had an unsaturated fatty acid at sn-1 and sn-2 positions (20:4-20:4>18:2-20:4>18:1-20:4). 16:0-20:4 and 18:0-20:4 PtdChos showed a minor specific activity. When labeled nuclei were reincubated in the absence of labeled substrate with the addition of cytosol, ATP and CoA, the specific activity of 20:4-20:4, 18:2-20:4 and 18:1-20:4 species decreased, while that of 16:0-20:4 and 18:0-20:4 increased. In conclusion, the asymmetric fatty acid distribution of saturated fatty acids at sn-1 position, and unsaturated fatty acids at sn-2 position of nuclear PtdCho molecular species was re-established by an acyl-CoA-dependent remodeling process.     

Effect of streptokinase on prostacyclin synthesis and phospholipase activity in cultured pulmonary artery endothelial cells

In this study, we examined the effects of streptokinase on arachidonic acid release and prostacyclin biosynthesis in cultured bovine pulmonary artery endothelial cells. When intact cells were incubated with streptokinase, a significant stimulatory effect on prostacyclin biosynthetic activity in cells was evident without any cellular damage at all concentrations used (1-10,000 units/ml). Streptokinase also caused a marked release of arachidonic acid. It induced rapid phospholipid hydrolysis, resulting in the release of up to 15% of incorporated [3H]arachidonic acid into the medium. After the addition of streptokinase, degradation of phosphatidylcholine and phosphatidylethanolamine was observed and lysophosphatidylcholine and lysophosphatidylethanolamine were produced. We also observed a transient rise in diacylglycerol after the addition of streptokinase. To test for phospholipase C activity, the release of incorporated [3H]choline, [3H]inositol and [3H]ethanolamine into the culture medium was determined. The level of radioactive inositol showed an increase, but the changes in choline and ethanolamine were comparatively small. An increase in inositol was detectable within 1 min after streptokinase addition and peaked after 15 min. Inositol phosphate and inositol trisphosphate were released, and these releases were suppressed by the addition of neomycin (50 microM). These results suggest that streptokinase stimulates phospholipase A2 and C activity, and that prostacyclin biosynthesis is subsequently increased in cultured endothelial cells.     

Selective incorporation of various C-22 polyunsaturated fatty acids in Ehrlich ascites tumor cells

Three 14C-labeled 22-carbon polyunsaturated fatty acids, 7,10,13,16-[14C]docosatetraenoic acid (22:4(n-6)), 7,10,13,16,19-[14C]docosapentaenoic acid (22:5(n-3)), and 4,7,10,13,16,19-[14C]docosahexaenoic acid (22:6(n-3)), were compared with [3H]arachidonic acid (20:4(n-6] and [14C]linoleic acid (18:2(n-6)) to characterize their incorporation into the lipids of Ehrlich ascites cells. The relatively rapid incorporation of the labeled 22-carbon acids into phosphatidic acid indicated that substantial amounts of these acids may be incorporated through the de novo pathway of phospholipid synthesis. In marked contrast to 20:4(n-6), the 22-carbon acids were incorporated much less into choline glycerophospholipids (CGP) and inositol glycerophospholipids (IGP). No selective preference was apparent for the (n-3) or (n-6) type of fatty acids. The amounts of the acids incorporated into diacylglycerophosphoethanolamine were in the order of: 22:6(n-3) greater than 20:4(n-6) much greater than 22:5(n-3) greater than or equal to 22:4(n-6) greater than 18:2(n-6), whereas for alkylacylglycerophosphoethanolamine they were in the order of: 22:4(n-6) greater than 22:6(n-3) greater than 22:5(n-3) much greater than 20:4(n-6) greater than 18:2(n-6). Of the mechanisms possibly responsible for the selective entry of 22-carbon acids into ethanolamine glycerophospholipids, the most reasonable explanation was that the cytidine-mediated ethanolamine phosphotransferase may have a unique double selectivity: for hexaenoic species of diacylglycerol and for 22-carbon polyunsaturated fatty acid-containing species of alkylacylglycerol. The relative distribution of fatty acids between newly incorporated and already maintained lipid classes suggested that IGP may function in Ehrlich cells as an intermediate pool for the retention of polyunsaturated fatty acids in glycerolipids.     

Selective changes of docosahexaenoic acid-containing phospholipid molecular species in monkey testis during puberty

Puberty has a profound effect upon the biochemical composition of the testis. We previously demonstrated that puberty was accompanied by great increases in the content of docosahexaenoic acid (DHA; 22:6 n-3) and dihomogamma-linoleic acid (20:3 n-6) and decreases in arachidonic acid (AA; 20:4 n-6) in the phospholipids of testis. In this report, we analyze the composition of the phospholipid molecular species of the ethanolamine and choline glycerophospholipids in the testis of prepubertal (2 years old) and young adult (7-8 years old) monkeys, There was an increase in the DHA species and a decrease in arachidonic species. Interestingly, with few exceptions, among the three molecules with DHA or AA at the sn-2 position, only 16:0-22:6 and 18:0-20:4 changed selectively in opposite directions for both ethanolamine and choline glycerophospholipids. In contrast, there was no such selectivity seen in molecular species containing dihomogamma-linoleic acid or linoleic acid at the sn-2 position. All three dihomogamma-linoleic acid species increased and all three linoleic acid species decreased during puberty. In summary, at puberty, i.e., the onset of spermatogenesis, there are selective changes in the phospholipid molecular species, particularly those containing DHA and AA. These changes suggest a specific functional role of DHA-containing molecular species in the lipid bilayer membranes of sperm cells. A possible link between the composition of DHA-phospholipid molecular species and cellular function is discussed.     

The biosynthesis of phosphatidylserines by acylation of 1-acyl-sn-glycero-3-phosphoserine in rat liver

The conversion of 1-[14C]acyl-sn-glycero-3-phosphoserine into molecular species of [14C]phosphatidylserine was studied using rat liver homogenate and microsomal preparations in the absence of added fatty acyl moieties. In liver homogenates, 81% of the newly-formed phosphatidylserines were tetraenoic (arachidonoyl) species while saturated, monoenoic, dienoic, trienoic, pentaenoic, and hexaenoic (docosahexaenoyl) species each represented 2-5% of the total. A similar pattern of molecular species was produced in liver microsomes. The selectivity of the microsomal acyl-CoA:1-acyl-sn-glycero-3-phosphoserine acyltransferase towards different acyl-CoA derivatives was also investigated. The relative suitability of the various acyl-CoA esters as substrates was found to be of the following order:20:4 = 18:2 greater than 18:1 greater than 16:0 = 18:0. These results with endogenous acyl donors suggest that the acylation of 1-acyl-sn-glycero-3-phosphoserine may partly account for the enrichment of liver phosphatidylserine in arachidonic acid but does not appear to be primarily responsible for the preponderance of docosahexaenoic acid in this phospholipid. The fatty acid specificity of the acyl-CoA: 1-acyl-sn-glycero-3-phosphoserine acyltransferase may contribute to the preferential formation of arachidonoyl phosphatidylserine.     

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.     

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.     

Bradykinin-stimulated release of [3H]arachidonic acid from phospholipids of HSDM1C1 cells: Comparison of diacyl phospholipids and plasmalogens as sources of prostaglandin precursors

Ethanolamine plasmalogens (1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamines) of many tissues contain high levels of arachidonate at their 2-position, and in certain tissues have been implicated as possible donors of arachidonate required in the synthesis of prostaglandins and thromboxanes. In the present study, [³H]arachidonate-labeled phospholipids of HSDM₁C₁ cells, a cell line derived from a mouse fibrosarcoma, were examined to determine the donor of the arachidonic acid released upon bradykinin stimulation of the synthesis of PGE₂. HSDM₁C₁ cells labeled with [³H]arachidonic acid for 24 hr in serum-free medium were used in most of the experiments and had the following distribution of label among the cellular lipids; phosphatidylcholine (33%), phosphatidylinositol (20%), diacyl-sn-glycero-3-phosphoethanolamine (15%), ethanolamine plasmalogen (15%), and less polar lipids (16%). Bradykinin treatment stimulated a rapid hydrolysis of [³H]arachidonate from the cellular lipids and conversion of the released acid to PGE₂, which was secreted into the medium. The label was released predominantly from phosphatidylinositol and possibly from phosphatidylcholine with no detectable change in the labeling of diacyl- or 1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. The ethanolamine plasmalogens, therefore, do not appear to be involved in the stimulated release of arachidonate in the HSDM₁C₁ cells. Indomethacin blocked the bradykinin-stimulated synthesis of PGE₂ and to a lesser degree inhibited the release of [³H]-arachidonate from the cellular lipids into the medium.     

Ethanolamine metabolism in cultured bovine aortic endothelial cells

The role of extracellular ethanolamine in phospholipid synthesis was examined in cultured bovine aortic endothelial cells. Serine and ethanolamine were both readily accumulated by these cells and incorporated into phospholipid. Exposing cells to extracellular ethanolamine for 4-6 weeks had no effect on cell growth, yet increased the phosphatidylethanolamine content of these cells by 31% as compared to control cells. The intracellular content of ethanolamine was measured by high performance liquid chromatography, and results showed that the ethanolamine-treated cells contained a significantly greater amount of free ethanolamine compared to control cells (0.62 +/- 0.07 nmol/mg of protein versus 0.27 +/- 0.05 nmol/mg of protein, respectively). Ethanolamine-treated cells also had decreased accumulation and incorporation into lipid of [3H]ethanolamine throughout a 48-h incubation and increased K'm and V'max parameters of ethanolamine transport as compared to control cells. Studies were also done to examine the effect of ethanolamine on the generation of free ethanolamine from phosphatidylserine. In pulse-chase experiments with [3H]serine, a physiological concentration of ethanolamine (25 microM) decreased the amount of 3H-labeled phosphatidylethanolamine produced from 3H-labeled phosphatidylserine by 12 h as compared to the amount of 3H-labeled phosphatidyl-ethanolamine produced in the absence of ethanolamine in the chase incubation. Furthermore, ethanolamine-treated cells accumulated 20% less labeled ethanolamine in the aqueous pool from [3H]serine after 24 h of incubation than did control cells. These results can be explained by isotope dilution with the ethanolamine pool that accumulates in these cells with time when exposed to media supplemented with a physiological concentration of ethanolamine and by an effect of ethanolamine on ethanolamine generation from phosphatidylserine. The results show that an extracellular source of ethanolamine significantly influences the phospholipid metabolism of cultured bovine aortic endothelial cells.     

Plasmalogen biosynthesis in Madin-Darby canine kidney cells: selectivity in the acylation of 1-alkyl-2-lyso-sn-glycero-3-phosphoethanolamine and the subsequent desaturation step

Acyl group specificity in the acylation of 1-alkyl-2-lyso-sn-glycero-3-phosphoethanolamine (1-alkyl-2-lyso-GroPEtn) to form 1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine (1-alkyl-2-acyl-GroPEtn) and the subsequent desaturation of 1-alkyl-2-acyl-GroPEtn to form plasmalogens (1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine, i.e., 1-alk-1'-enyl-2-acyl-GroPEtn) was investigated in intact Madin-Darby canine kidney (MDCK) cells and cell-free membrane preparations. We found 1-[3H]alkyl-2-lyso-GroPEtn was selectively acylated with polyunsaturated fatty acids in the order 20:4 greater than 20:5 greater than 20:3 (n-9) greater than 22:6 by cell-free membrane preparations of MDCK cells. The same pattern of acyl specificity was seen in intact MDCK cells, although the intact cells produced significantly larger amounts of 1-[3H]alkyl-2-acyl-GroPEtn containing oleic acid. There was an increased desaturation of the 1-[3H]alkyl-2-acyl-GroPEtn species containing docosahexaenoic acid to plasmalogens (1-[3H]alk-1'-enyl-2-acyl-GroPEtn) by both intact MDCK cells and the cell-free membrane preparations. The relatively rapid disappearance of the 1-[3H]alk-1'-enyl-2-docosahexaenoyl-GroPEtn species during a 20-h incubation of prelabeled intact MDCK cells suggests a more rapid turnover of this molecular species. Our results indicate there is a high selectivity in the final acylation and desaturation steps of the biosynthetic pathway for plasmalogens.