Arachidonate cannot be released directly from diacyl-sn-glycero-3-phosphocholine in thrombin-stimulated platelets.

The origin of the arachidonate released from platelets on stimulation with thrombin was investigated by comparing the specific activities of released arachidonate and of arachidonoyl-containing phospholipids using rat platelets prelabelled with arachidonate. Quantification of the released arachidonate was determined in the presence of BW 755 C, a dual cyclo-oxygenase/lipoxygenase inhibitor, which was found not to modify the arachidonate mobilization between the platelet phospholipids. The phospholipid molecular species were analysed by h.p.l.c. of diradylglycerol benzoate derivatives of diacyl, alkylacyl and alkenylacyl classes. The labelled/unlabelled arachidonate ratio varied greatly in the phospholipids depending on whether an ether or acyl bond was present in sn-1 position of the glycerol, on the length and degree of unsaturation of this fatty chain and on the polar head group. Between 15 s and 5 min of stimulation by thrombin, the released arachidonate kept a constant specific activity which was considerably lower than the specific activity of diacyl-GPC. The specific activity of the released arachidonate was intermediate between the specific activities of the 16:0-20:4 and 18:0-20:4 species of diacyl-GPI and diacyl-GPE, and corresponded to the mean specific activity of alkylacyl-GPC. The data indicate that the released arachidonate cannot come directly from diacyl-GPC, and that two phospholipids in particular can act as direct precursors of the released arachidonate. These are (1) the alkylacyl-GPC and (2) the diacyl-GPE whose hydrolysis would induce an arachidonate transfer from diacyl-GPC.     

Changes in fatty chain compositions of lipid classes during frozen storage of the adductor muscle of giant ezo scallop (Patinopecten yessoensis)

Changes in lipid components, particularly glycerophospholipids in the adductor muscle of giant ezo scallop during storage at −20°C, were investigated. During storage, the contents of total lipid (TL) and polar lipid (PL) decreased but that of non-polar lipid (NL) increased. Glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE) decreased by 50 and 15% of the each initial content, while lyso-GPC and free fatty acids increased. The percentages of polyunsaturated fatty acids such as 20:5n-3 and 22:6n-3 in the TL and PL fractions decreased during storage, but those of the polyunsaturated fatty acids in the NL increased. In the alkenylacyl-GPE and diacyl-GPC, the percentages of relatively longer acids in the sn-1 positions of glycerol moieties decreased at higher rates than did the shorter chains, whereas the results for diacyl-GPE were opposite to those of alkenylacyl-GPE and diacyl-GPC. In the prominent fatty acids in the sn-2 positions of alkenylacyl-GPE and diacyl-GPC, the percentage of 22:6n-3 decreased from compared to the high level of 20:5n-3, while that of diacyl-GPE increased.     

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.     

Electrospray ionization mass spectrometric analyses of phospholipids from INS-1 insulinoma cells: comparison to pancreatic islets and effects of fatty acid supplementation on phospholipid composition and insulin secretion

Insulin secretion by pancreatic islet beta-cells is impaired in diabetes mellitus, and normal beta-cells are enriched in phospholipids with arachidonate as sn-2 substituent. Such molecules may play structural roles in exocytotic membrane fusion or serve as substrates for phospholipases activated by insulin secretagogues. INS-1 insulinoma cells respond to secretagogues and permit the study of effects of culture with free fatty acids on phospholipid composition and secretion. INS-1 cell glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) lipids are demonstrated here by electrospray ionization mass spectrometry to contain a lower fraction of molecules with arachidonate and a higher fraction with oleate as sn-2 substituent than native islets. Palmitic acid supplementation induces little change in these INS-1 cell lipids, but supplementation with linoleate or arachidonate induces a large rise in the fraction of INS-1 cell GPC species with polyunsaturated sn-2 substituents and a fall in oleate-containing species to yield a GPC profile similar to native islets. The fraction of GPE lipids comprised of plasmenylethanolamine species with polyunsaturated sn-2 substituents in early-passage INS-1 cells is similar to that of islets, but declines on serial passage. Such molecules might participate in exocytotic membrane fusion, and late-passage INS-1 cells have reduced insulin secretory responses. Arachidonate supplementation induces a rise in the fraction of INS-1 cell GPE lipids with polyunsaturated sn-2 substituents and partially restores responses to insulin secretagogues by late-passage INS-1 cells, but does not further amplify secretion by early-passage cells. Effects of extracellular free fatty acids on beta-cell phospholipid composition and secretory responses could be involved in changes in beta-cell function during the period of hyper-free fatty acidemia that precedes diabetes mellitus.     

Effects of postdecapitative ischemia and hypoxia on the phosphoglyceride acyl groups of rat brain membranes

Rats subjected to mild hypoxic and postdecapitative ischemic treatments indicated a decrease (8–16%) in the proportion of polyunsaturated acyl groups of diacyl glycerophosphocholines (diacyl-GPC), diacyl glycerophosphoethanolamines (diacyl-GPE), and alkenylacyl glycerophosphoethanolamines (alkenylacyl-GPE) in brain synaptosomes. In general, the acyl group changes due to mild hypoxic treatment were less obvious than those due to the ischemic treatment. The decrease in polyunsaturated acyl groups was marked by an increase in the saturated (16:0 and 18:0) and monoenoic (18:1) acyl groups. Among the polyunsaturated acyl groups, arachidonate (20:4) indicated the greatest decrease in response to ischemic and hypoxic treatments. The decrease in polyunsaturated fatty acids of diacyl glycerophosphocholines was largest in the first minute of ischemic treatment and the first 30 min of hypoxic treatment. After the initial decrease, there was a slight recovery. The biphasic type of change was thought to be due to active reacylation of the lyso phospholipids. This biphasic change, however, was not observed with ethanolamine phosphoglycerides which indicated a steady decrease in the polyunsaturated acyl group content with time of ischemic treatment. The increased hydrolysis of polyunsaturated acyl groups in brain membrane phosphoglycerides due to the ischemic and hypoxic treatments seemed to correlate well with the implication of phospholipase A₂ involvement in eliciting the increase in free fatty acids during brain stimulation.     

Aldehydic peptides inhibiting renin

The metabolism of 20:4 (arachidonic acid) in alkenylacyl, alkylacyl and diacyl lipid classes in choline glycerophospholipids (CGP) and ethanolamine glycerophospholipids (EGP) in rabbit alveolar macrophages was examined. [3H]20:4 was very rapidly incorporated into diacyl glycerophosphocholine (GPC). After the removal of free 20:4, the radioactivity was gradually lost from diacyl GPC. Concomitantly, the radioactivities in alkylacyl GPC and alkenylacyl glycerophosphoethanolamine (GPE) were increased, indicating that 20:4 was mobilized from diacyl GPC to alkylacyl GPC and alkenylacyl GPE. The mobilization was considered to be a 20:4-specific event. The gradual accumulation of 20:4 in ether phospholipids leads to a high abundance of 20:4 in these lipids. These results suggest metabolic relationships between 20:4 and ether phospholipids, including platelet-activating factor (PAF).     

CHANGES IN ACYL GROUP COMPOSITION OF DIACYL-GLYCEROPHOSPHORYLETHANOLAMINE, ALKENYLACYL-GLYCEROPHOSPHORYLETHANOLAMINE AND DIACYL-GLYCEROPHOSPHORYLCHOLINE IN MYELIN AND MICROSOMAL FRACTIONS OF MOUSE BRAIN DURING DEVELOPMENT

—Age-related changes in acyl group composition of diacyl-glycerophosphorylethanolamine (GPE), alkenylacyl-GPE and diacyl-glycerophosphorylcholine (GPC) were examined in myelin and microsomal fractions of mouse brain during development. In general, the phosphoglycerides in the myelin fraction showed an increase in the proportions of 18:1 and 20:1 and a decrease in the proportions of 16:0, 20:4(n-6) and 22:6(n-3) with increasing age. These changes were especially obvious with the acyl groups of alkenylacyl-GPE. The acyl group profiles of phosphoglycerides in the microsomal fraction were different from those in the myelin fraction. During development, there was an increase in 22:6 and a decrease in 20:4 in the phosphoglycerides of microsomes. These changes were especially obvious with the diacyl-GPE. Starting from 2 weeks of age, there was also an increase in the proportions of 18:1 and 20:1 in alkenylacyl-GPE in the microsomal fraction but this change was not as dramatic as that in the myelin fraction. In general, the acyl groups of diacyl-GPC in both myelin and microsomal fractions showed only little age-related changes as compared to the ethanolamine phosphoglycerides. Results suggest an induction in the synthesis of monoenoic fatty acids in brain during development. The monoenoic fatty acids synthesized during this period are rapidly and preferentially incorporated into the ethanolamine plasmalogen for further utilization in synthesis of the myelin membranes.     

Arachidonate mobilization in diacyl, alkylacyl and alkenylacyl phospholipids on stimulation of rat platelets by thrombin and the Ca2+ ionophore A23187.

Platelet stimulation by thrombin or Ca2+ ionophore induces mobilization of arachidonate from lipid stores. We have previously shown that, in [14C]arachidonic acid-prelabelled resting platelets, [14C]arachidonate was transferred from diacyl-sn-glycerophosphocholine to ethanolamine and choline-containing ether phospholipids. This transfer reached an equilibrium after 5 h incubation [Colard, Breton & Bereziat (1984a) Biochem. J. 222, 657-662]. [14C]Arachidonate-prelabelled platelets having reached this transfer equilibrium were used to study the mobilization of arachidonate in etheracyl and diacyl phospholipids. Upon thrombin stimulation, arachidonate decreased in diacyl-sn-glycero-3-phosphoinositol, in alkylacyl- and diacyl-sn-glycero-3-phosphocholine and increased in alkenylacyl- and diacyl-sn-glycero-3-phosphoethanolamine. Upon challenge with Ca2+ ionophore A23187, arachidonate decreased in diacyl-sn-glycero-3-phosphoethanolamine, in diacyl- and alkylacyl-sn-glycero-3-phosphocholine and increased in alkenylacyl-sn-glycero-3-phosphoethanolamine. We also compared arachidonate mobilization in platelets stimulated immediately after [14C]arachidonic acid chase with platelets stimulated after 5 h reincubation. We observed that the arachidonate newly incorporated into diacyl-sn-glycero-3-phosphocholine and triacylglycerols was rapidly released upon stimulation. This suggests the presence in these two lipids of a rapidly-turning-over arachidonate pool.     

Turnover Rates of the Molecular Species of Ethanolamine Plasmalogen of Rat Brain

1,2-Diradyl-3-acetylglycerols prepared from 1-O-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (alkenylacyl-GPE, ethanolamine plasmalogen) and 1-alkyl-2-acylglycero-3-phosphoethanolamine (alkylacyl-GPE) of rat brain at 18 days of age were subfractionated into six species by AgNO3-impregnated TLC. The percent compositions of substractions were compared with that of 1,2-diacylglycero-3-phosphoethanolamine (diacyl-GPE). The incorporation rate of [1-3H]glycerol into each molecular species was also estimated to examine the turnover rate and selective synthesis of molecular species of ethanolamine phosphoglycerides (EPG). Among the molecular species of EPG, a major proportion contained polyunsaturated fatty chains, and the sum of tetraene-, pentaene-, and hexaene-containing species was greater than 65% in common with three classes of EPG. It was possible to calculate the turnover time, synthesis rate, and synthesis rate constant of ethanolamine plasmalogen in myelinating rat brain by the equation of Zilversmit et al. since the time-dependent change of specific activity and the distribution of molecular species indicated that each molecular species of alkenylacyl-GPE is synthesized from the corresponding species of alkylacyl-GPE. The observed turnover time of ethanolamine plasmalogen was about 5 h. The observed turnover times of the various molecular species were of the order: tetraene greater than or equal to hexaene greater than pentaene greater than or equal to monoene greater than or equal to diene. The synthesis rate constants of each molecular species, in the formation of alkenylacyl-GPE from alkylacyl-GPE, were of the order: hexaene greater than tetraene greater than pentaene greater than diene greater than or equal to monoene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of alkyl and alkenyl ether-linked phospholipids and platelet-activating factor-like lipid in various species of invertebrates.

The levels of alkenylacyl, alkylacyl and diacyl subclasses of choline glycerophospholipid (CGP) and ethanolamine glycerophospholipid (EGP) fractions in 28 species of various invertebrates were studied. We found that only small amounts of either 1-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) or 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine (alkenylacyl-GPE) are present in most species of insects. On the other hand, almost all species examined in various phyla other than Arthropoda were shown to contain large amounts of both alkylacyl-GPC and alkenylacyl-GPE. The highest proportion of alkylacyl subclass in CGP was noted in sponge, Halichondria japonica (81.8% of CGP) and the highest proportion of alkenylacyl subclass in EGP was found in clam worm, Marphysa sanguinea (88.7% of EGP). We next surveyed the presence of platelet-activating factor (PAF)-like lipid in 45 species of invertebrates. PAF-like lipid was widely distributed among various lower animals. The highest value was obtained for sea cucumber, Stichopus japonicus, in which PAF-like lipid was present throughout the body. We also confirmed the presence of acetyltransferase activity in several lower animals. These results suggest that alkyl and alkenyl ether-linked phospholipids including PAF are physiologically important molecules particularly for invertebrates belonging to lower phyla.     

Release of arachidonic acid from 1-alkyl-2-acyl-sn-glycero-3-phosphocholine, a precursor of platelet-activating factor, in rat alveolar macrophages

Platelet activating factor and the bioactive metabolites of arachidonic acid are secreted by alveolar macrophages in response to stimulation by phagocytic agents or calcium ionophore. We have previously shown a deacylation-acetylation sequence in the formation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) from alkylacyl-(long chain)-GPC (Albert, D.H. and Snyder, F. (1983) J. Biol. Chem. 258, 97-102). This sequence may be an important source of 20:4 during inflammatory reactions since, in alveolar macrophages, the ether lipid precursor of PAF represents 35% of the choline glycerophospholipids and has a much higher content (35%) of 20:4 in the sn-2 position than does diacyl-GPC (17%). Alveolar macrophages prelabeled with 14C-labeled fatty acids (16:0, 18:1, 18:2 and 20:4) and [1-3H]alkyllyso-GPC were used to study the release of fatty acids from ether-linked and diacyl phospholipids. Each of these fatty acids was incorporated primarily into the choline glycerophospholipids of alveolar macrophages. The release of 20:4 from macrophage phospholipids was increased by treatment of the labeled cells with the calcium ionophore A23187 (2 microM) or zymosan (1 mg/ml), whereas the release of 16:0, 18:1 and 18:2 was not increased above control levels by either stimuli. Although more of the labeled 20:4 is released from the diacyl-GPC (50% of the total released), substantial amounts (44%) of 20:4 are derived from alkylacyl-GPC after incubating the stimulated cells for 60 min. The loss of 20:4 continued from the diacyl species throughout the incubation period studied, whereas a slower net release of 20:4 lost from the alkylacyl-GPC fraction was evident after 2 h. We conclude that the deacylation-reacylation cycle is an important aspect of the metabolism of 20:4 and alkylacyl-GPC during inflammatory stimulation of alveolar macrophages and that the deacylation of this ether-linked phospholipid (which is the first step in the formation of PAF) is responsible for a significant amount of the 20:4 released.     

Transacylation of 1-O-alkyl-SN-glycero-3-phosphocholine (lyso platelet-activating factor) and 1-O-alkenyl-SN-glycero-3-phosphoethanolamine with docosahexaenoic acid (22:6 omega 3)

[14C]22:6 (docosahexaenoic acid) was rapidly incorporated into cellular lipids in rabbit alveolar macrophages. After removal of free [14C]22:6, the radioactivity in diacyl-glycerophosphocholine (GPC) gradually decreased with a concomitant increase in [14C]22:6 in alkylacyl-GPC and alkenylacyl-glycerophosphoethanolamine (GPE), indicating that [14C]22:6 was transferred from diacyl-GPC to these ether lipid fractions. In fact, macrophage microsomes were shown to catalyze the transfer of [14C]22:6 from exogenously added diacyl-GPC to 1-alkyl-GPC (lyso platelet-activating factor) and 1-alkenyl-GPE. These results are the first evidence for the involvement of the transacylation system in the metabolism of C22 polyunsaturated fatty acids and lyso platelet-activating factor.     

SYNTHESIS OF ETHANOLAMINE PHOSPHOGLYCERIDES BY MICROSOMES FROM THE BRAINS OF JIMPY AND QUAKING MICE

Abstract— —Brains of jimpy and quaking mice are known to be deficient in myelin and alkenylacyl-glycero-phosphorylethanolamines (alkenylacyl-GPE, ethanolamine plasmalogens). Ethanolamine plasmalogen synthetic activity appeared to be normal and ethanolamine phosphotransferase (EC 2.7.8.1) activities are higher in the brain microsomes from jimpy and quaking mice than in their littermate controls when the activities are assayed with alkylacylglycerols and CDP[¹⁴C]ethanolamine. When endogenous diradylglycerols were the substrate, the rate of synthesis of diacyl-GPE was normal but the rate of synthesis of the ether lipids, alkenylacyl-GPE and alkylacyl-GPE, was 33% and 8% below control levels for jimpy brain microsomes and quaking brain microsomes respectively. This difference is probably due to a normal content of diacylglycerols and a deficient content of alkylacylglycerols in the mutant brain microsomes. The apparent alkylacylglycerol deficiencies in the microsomes correspond with the ethanolamine plasmalogen deficiencies in the brains of these mutant mice.     

Molecular species of phospholipid in rats in primary and transplanted fibrosarcomas induced by soybean oil containing tocopherol acetate.

When soybean oil containing tocopherol acetate was given to rats once a week subcutaneously for 10-12 months, it caused the development of fibrosarcomas at the injection site in 11 of 15 rats. A tumor produced in this manner proved eminently transplantable into other rats. The molecular species of phospholipid subclasses were determined in primary and transplanted tumors. The molecular species composition of the phospholipid subclasses in both types of tumors were similar. The percentages of diacyl and alkylacyl glycerophosphocholine (GPC) were 90-93 and 6-8% of total phosphatidylcholine, respectively. The percentages of diacyl and alkenylacyl glycerophosphoethanolamine (GPE) were 51 and 45%, respectively, of total phosphatidylethanolamine (PE). Diacyl and alkylacyl GPC species containing arachidonic acid (20:4) composed about 15-16 and 37-40% of each subclass, respectively. Diacyl and alkenylacyl GPE species containing 20:4 composed about 38-40 and 56-60% of each subclass, respectively. Disaturated species of diacyl and alkylacyl GPC composed about 22-24 and 13% of each subclass, respectively, whereas these species of PE composed less than 2%. The fatty acid composition of the other tumor phospholipids was analyzed.     

Effect of incubation of guinea-pig taenia coli in potassium-free media on arachidonate release and lipid metabolism

We studied the effects of immersion of guinea-pig taenia coli strips in potassium-free media on arachidonate stores and other lipid fractions. Control studies obtained with the strips in Krebs solution showed that greater than 97% of arachidonate was found esterified in phospholipid with the following distribution: phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylserine plus phosphatidylinositol. 30 min incubation of the strips with [3H]arachidonate complexed to albumin resulted in incorporation of this isotope into phospholipid and neutral lipid fractions, phosphatidylcholine greater than neutral lipid greater than phosphatidylserine plus phosphatidylinositol greater than phosphatidylethanolamine. 30 min incubations with 32PO4(2-)-resulted in an isotope incorporation into phospholipids, phosphatidylcholine greater than phosphatidylserine plus phosphatidylinositol greater than phosphatidylethanolamine. After 'loading' with [3H]arachidonate and 32P, placing the strips in potassium-free media caused the following: there was an increased release of [3H]arachidonate from the tissue into the bathing solution. [3H]Arachidonate and 32P radioactivity in phosphatidylinositol fell without a change in phosphatidylinositol content. [3H]Arachidonate and 32P radioactivity in other phospholipid fractions was unchanged. Arachidonate specific activity fell and arachidonate content increased in the phosphatidylserine plus phosphatidylinositol fraction. [3]Arachidonate in neutral lipid did not change significantly. We conclude that exposure of taenia coli to potassium-free media activates turnover of phosphatidylinositol, which results in release of arachidonate.     

Acyl group composition of membrane phospholipids in mammary tissues and carcinoma induced by dimethylbenz(a)anthracene.

Phospholipids and their acyl group composition in mammary adenocarcinomas and mammary tissue of the same tumor-bearing animals were investigated. Breast adenocarcinoma induced by dimethybenz(a)anthracene exhibited a phospholipid pattern which was different from that of the mammary tissue. Tumor phospholipids had higher proportions of diacyl-GPI, diacyl-GPE, and alkenylacyl-GPE and a lower proportion of diacyl-GPC than the controls. The acyl groups of most phospholipids in tumors showed a marked increase in the proportion of 18:1 and a decrease in the proportion of 18:2. The fatty acid composition of plasmalogen and triglyceride, however, remained unchanged. In spite of the decrease in the proportion of 18:2, there was no apparent difference in the proportion of 20:4 in most of the phosphoglycerides; however, a significant decrease in this fatty acid was noted in diacyl-GPI. Results of this study demonstrated that the membrane phospholipids of mammary adenocarcinoma were altered in respect to acyl group composition. Changes in physical properties of the cell membrane, in turn, could lead to abnormal manifestation of membrane regulated events in tumor cells.     

Phospholipid remodeling in human neutrophils. Parallel activation of a deacylation/reacylation cycle and platelet-activating factor synthesis

A23187 stimulated two enzymatic activities of human neutrophils (polymorphonuclear leukocytes), phospholipase A2 and fatty acyl-CoA acyltransferase, which resulted in a stimulated deacylation/reacylation cycle. The incorporation of fatty acids, other than arachidonic or eicosapentaenoic acid, into diacyl and alkylacyl species of choline phosphoglycerides was stimulated by 10-fold by A23187. These fatty acids were exclusively incorporated into the sn-2 position, and [3H]glycerol labeling showed there was no stimulation of de novo synthesis. A23187 also stimulated fatty acid incorporation into other phospholipids, but de novo synthesis accounted for a portion of this uptake. Inhibitors of protein kinase C prevented the stimulated recycling of phosphatidylcholine, and the simultaneous induction of platelet-activating factor synthesis, by inhibiting phospholipase A2 activation. They inhibited [3H]arachidonate release from prelabeled polymorphonuclear leukocytes, but had no effect on in vitro fatty acyl-CoA acyltransferase or acetyl-CoA acetyltransferase activity. Extracts from A23187-treated cells contained a fatty acyl-CoA acyltransferase, which did not utilize arachidonoyl-CoA, that was 2.3-fold more active than that of control extracts. Phosphatase treatment decreased this stimulated activity by 66%. Thus, A23187 stimulated a phospholipase A2 activity that generated both 1-alkyl and 1-acyl lysophosphatidylcholines. A stimulated acetyltransferase used a portion of the alkyl species for platelet-activating factor synthesis, while the acyl species and residual alkyl species were rapidly reacylated to phosphatidylcholine by a stimulated acyl-transferase. Arachidonate, an eicosanoid precursor, was spared by this process.     

Fatty acid composition of diacyl, alkylacyl, and alkenylacyl phospholipids of control and arachidonate-depleted rat polymorphonuclear leukocytes

Phospholipid fatty acid composition and phospholipid subclass distribution of control and arachidonate-depleted rat polymorphonuclear leukocytes (PMN) were compared. The 20:4-depleted PMN contained significantly higher amounts of 16:1, 18:1 and 20:3 (delta 5,8,11) and lower amounts of 18:2 and 20:4 than the phospholipids from control cells. Choline-containing glycerophospholipids (CGP) were the major phospholipids of both control and 20:4-depleted cells representing 34% and 37% of the total phospholipids, respectively. Significant amounts of ethanolamine-containing glycerophospholipids (EGP) (29% and 30%) and sphingolipids (20% and 18%) were also present in both cell types. Serine-containing glycerophospholipids (SGP) together with inositol-containing glycerophospholipids (IGP) constituted 16% and 13% of the phospholipids in control and 20:4-depleted cells, respectively. CGP from control cells had significantly higher amounts of 16:0 and 18:2 and lower amounts of 18:0 and 20:4 than EGP, whereas CGP from 20:4-depleted cells has higher amounts of 16:0 and 16:1 and lower amounts of 20:3 than EGP. Analysis of the subclass composition of CGP and EGP revealed that both control and 20:4-depleted cells contained significantly large amounts of alkylacyl-GPC and alkenylacyl-GPE. Small amounts of alkylacyl-GPE and alkenylacyl-GPC were also observed. The predominant fatty acyl residues found in the 1,2-diacyl-GPC, alkylacyl-GPC of control cells were 16:0, 18:0, 18:1, 18:2, and 20:4, while those of 20:4-depleted cells were 16:0, 16:1, 18:0, 18:1, and 20:3. More than 60% of CGP-bound 20:4 of control cells and about 70% of the CGP-bound 20:3 of 20:4-depleted cells were found in their alkylacyl species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of the role of group VI phospholipase A2 in fatty acid incorporation, phospholipid remodeling, lysophosphatidylcholine generation, and secretagogue-induced arachidonic acid release in pancreatic islets and insulinoma cells.

An 84-kDa group VI phospholipase A2 (iPLA2) that does not require Ca2+ for catalysis has been cloned from Chinese hamster ovary cells, murine P388D1 cells, and pancreatic islet beta-cells. A housekeeping role for iPLA2 in generating lysophosphatidylcholine (LPC) acceptors for arachidonic acid incorporation into phosphatidylcholine (PC) has been proposed because iPLA2 inhibition reduces LPC levels and suppresses arachidonate incorporation and phospholipid remodeling in P388D1 cells. Because islet beta-cell phospholipids are enriched in arachidonate, we have examined the role of iPLA2 in arachidonate incorporation into islets and INS-1 insulinoma cells. Inhibition of iPLA2 with a bromoenol lactone (BEL) suicide substrate did not suppress and generally enhanced [3H]arachidonate incorporation into these cells in the presence or absence of extracellular calcium at varied time points and BEL concentrations. Arachidonate incorporation into islet phospholipids involved deacylation-reacylation and not de novo synthesis, as indicated by experiments with varied extracellular glucose concentrations and by examining [14C]glucose incorporation into phospholipids. BEL also inhibited islet cytosolic phosphatidate phosphohydrolase (PAPH), but the PAPH inhibitor propranolol did not affect arachidonate incorporation into islet or INS-1 cell phospholipids. Inhibition of islet iPLA2 did not alter the phospholipid head-group classes into which [3H]arachidonate was initially incorporated or its subsequent transfer from PC to other lipids. Electrospray ionization mass spectrometric measurements indicated that inhibition of INS-1 cell iPLA2 accelerated arachidonate incorporation into PC and that inhibition of islet iPLA2 reduced LPC levels by 25%, suggesting that LPC mass does not limit arachidonate incorporation into islet PC. Gas chromatography/mass spectrometry measurements indicated that BEL but not propranolol suppressed insulin secretagogue-induced hydrolysis of arachidonate from islet phospholipids. In islets and INS-1 cells, iPLA2 is thus not required for arachidonate incorporation or phospholipid remodeling and may play other roles in these cells.     

Ca2+ effect on ATP-independent lysophospholipid transacylases is indissociable from Ca2+ effect on phospholipase A2 activity in rat platelet sonicates

CoA-dependent transacylation and phospholipid hydrolysis were studied in parallel experiments using rat platelet sonicates. The decrease observed in palmitoyllyso-sn-glycero-3-phosphocholine (palmitoyllyso-GPC) transcylation as a function of Ca2+ concentration was found to be correlated with appearance of endogenous lysoderivatives. We also demonstrated that endogenously produced acyllyso-sn-glycero-3-phosphoethanolamine (acyllyso-GPE) induced CoA-dependent arachidonate transfer from diacyl-GPC. These results further argue for a two-step arachidonate release from diacyl-GPC when platelets are stimulated with thrombin.