Phospholipid metabolism, calcium flux, and the receptor-mediated induction of chemotaxis in rabbit neutrophils

Rabbit neutrophils were stimulated with the chemotactic peptide fMet-Leu-Phe in the presence of the methyltransferase inhibitors homocysteine (HCYS) and 3-deazaadenosine (3-DZA). HCYS and 3-DZA inhibited chemotaxis, phospholipid methylation, and protein carboxymethylation in a dose-dependent manner. The chemotactic peptide-stimulated release of [14C]arachidonic acid previously incorporated into phospholipid was also partially blocked by the methyltransferase inhibitors. Stimulation by fMet-Leu-Phe or the calcium ionophore A23187 caused release of arachidonic acid but not of previously incorporated [14C]-labeled linoleic, oleic, or stearic acids. Unlike the arachidonic acid release caused by fMet-Leu-Phe, release stimulated by the ionophore could not be inhibited by HCYS and 3-DZA, suggesting that the release was caused by a different mechanism or by stimulating a step after methylation in the pathway from receptor activation to arachidonic acid release. Extracellular calcium was required for arachidonic acid release, and methyltransferase inhibitors were found to partially inhibit chemotactic peptide-stimulated calcium influx. These results suggest that methylation pathways may be associated with the chemotactic peptide receptor stimulation of calcium influx and activation of a phospholipase A2 specific for cleaving arachidonic acid from phospholipids.     

Patterns of fatty acid release from endogenous substrates by human platelet homogenates and membranes.

We describe a method for measuring the release of fatty acids from endogenous substrates of human platelet homogenates and membranes. The method depends on the availability of lipids whose fatty acids are odd-chained and therefore suitable as internal reference compounds that, at the time of lipid extraction, can be added to an incubation to permit subsequent quantification of the content of free fatty acids or fatty acids esterified to specific lipids. We found four types of lipolytic activities in human platelets. In homogenates at pH 4.0 a triglyceride lipase operated as shown by the synchrony of triglyceride degradation and release of glycerol and those fatty acids that are the predominant constituents of triglycerides. However, enough arachidonic acid was released at this pH level to suggest some phospholipid breakdown, since triglycerides hold relatively small amounts of this acid. With membranous preparations, in the alkaline pH range there were two peaks of fatty acid release with accompanying degradation of phospholipids. At pH 8.5, where release of the saturated acids, palmitic and stearic, predominated, their sum was 3.5 times that of arachidonic acid. At pH 9.5 the release of palmitic and stearic acids was only slightly below their peak values; however, the release of arachidonic acid nearly equaled the sum of the saturated acids. Linoleic acid was not released in representative amounts by those reactions that released arachidonic acid, despite the overwhelming propensity of both to be esterified at the 2-position of phospholipids. Pertinently, the choline phospholipids are linoleic-rich and the non-choline phospholipids linoleic-poor, while both have a generous endowment of arachidonic acid. With this in mind, we raise the possibility that the phospholipase A2 of human platelets is an endoenzyme because of its tendency to act on those phospholipids that are thought to comprise the inner layer of the cell membrane.     

Effect of leukotriene B4, prostaglandin E2 and arachidonic acid on cytosolic-free calcium in human neutrophils

Changes in cytosolic free calcium [Ca2+]i and release of beta-glucuronidase in response to leukotriene B4 (LTB4) were measured in intact neutrophils loaded with the fluorescent Ca2+ indicator, quin 2. LTB4 (10(-10) M or higher) caused a rapid rise in [Ca2+]i due to influx from the extracellular medium and release from intracellular pools as well as enzyme release. PGE2 (3 microM) did not alter [Ca2+]i whereas arachidonic acid (10 microM) raised [Ca2+]i. Pretreatment of cells with the chemotactic peptide FMLP inhibited the subsequent rise of [Ca2+]i induced by LTB4. Since chemotactic peptides activate the lipoxygenase pathway of arachidonic acid metabolism, it may be speculated that endogenous LTB4 generation is involved in neutrophil activation.     

Evidence for differential activation of arachidonic acid metabolism in formylpeptide- and macrophage-activation-factor-stimulated guinea-pig macrophages.

Alterations of phospholipid and arachidonic acid metabolism were studied by treatment of guinea-pig peritoneal-exudate macrophages with chemotactic peptide, formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) and macrophage activation factor (MAF). The chemotactic peptide caused a rapid rearrangement in inositol phospholipids, including a breakdown of polyphosphoinositides within 30s, followed by a resultant formation of phosphatidylinositol (PI), diacylglycerol, phosphatidic acid and non-esterified arachidonic acid within 5 min. In addition to these sequential alterations, arachidonic acid was released mainly from PI. On the other hand, MAF induced a slow liberation of arachidonic acid, mainly from phosphatidylethanolamine (PE) and phosphatidylcholine (PC) by phospholipase A2 after the incubation period of 30 min, but not any rapid changes in phospholipids. Treatment of macrophages for 15 min with fMet-Leu-Phe produced the leukotrienes (LTs) B4, C4 and D4, prostaglandins (PG) E2 and F2 alpha and thromboxane (TX) B2. In contrast, MAF could not stimulate the production of arachidonic acid metabolites during the incubation period of 15 min, but could enhance that of PGE2, PGF2 alpha, TXB2 and hydroxyeicosatetraenoic acids at 6 h. However, the stimulated formation of LTs was not detected at any time. These results indicate that the effects of fMet-Leu-Phe on both phospholipid and arachidonic acid metabolism are very different from those mediated by MAF.     

Phospholipase A2-mediated release of arachidonic acid in stimulated guinea pig alveolar macrophages: interaction with lipid mediators and cyclic AMP

The stimulation of cultured guinea pig alveolar macrophages by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine, or by the phospholipid inflammatory mediator platelet activating factor (PAF) induced an increase in arachidonic acid release and its cyclooxygenase products. This release, which was mimicked by the association of threshold concentrations of the calcium ionophore A 23187 and of the protein kinase C activator tetradecanoyl phorbol acetate arose mainly from diacyl- and alkyl-acyl-phosphatidylcholine and phosphatidylinositol. Using [1-14C]arachidonic acid-labeled membranes as an endogenous substrate as well as dioleoyl-phosphatidyl [14C]ethanolamine as an exogenous substrate, we showed that phospholipase A2 activity of stimulated macrophages increases upon stimulation. Treatment of macrophages by prostaglandin E2 decreased the arachidonic acid release elicited by the chemotactic peptide and PAF. Furthermore, prostaglandin E2 increased and PAF decreased the cellular content in cyclic AMP. From these results we suggest that an initial stimulation of alveolar macrophages by a bacterial signal initiates the sequential activation of a phospholipase C and of phospholipase A2, leading to the release of PAF and eicosanoids. These mediators may in turn modulate the cell response by increasing or decreasing cyclic AMP, Ca2+, or diacyglycerol macrophage content.     

12-L-hydroxy-5,8,10,14-eicosatetraenoic acid, a chemotactic fatty acid, is incorporated into neutrophil phospholipids and triglyceride.

Platelets contain a lipoxygenase which converts arachidonic acid to 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) which has been shown to be chemotactic for human neutrophils and eosinophils. [14C]-12-HETE was biosynthesized, purified and incubated at a concentration of 1 micro M with human neutrophils. Lipids were extracted from the neutrophils and the media, and the radiolabeled products identified. 26 percent of the radiolabel was found in the cells after 30 min incubation, essentially all of it esterified into phospholipid and triglyceride. The radiolabeled phospholipids and triglycerides were transesterified and the liberated fatty acid was identified as [14C]-12-HETE. This is the first demonstration of direct alteration of membrane components by a chemotactic agent and may be an example of a more generalized mechanism for altering membrane characteristics.     

Efflux of gamma-aminobutyric acid from and appearance of free arachidonic acid inside synaptosomes

When synaptosomes were depolarized in the presence of Ca2+, or when Ca2+ was added to synaptosomes pretreated with Ca2+ ionophore (A23187), free arachidonic acid was clearly increased within synaptosomes, and at the same time an efflux of gamma-aminobutyric acid from synaptosomes was observed. Moreover, when synaptosomes labelled with [14C]arachidonic acid were depolarized in the presence of Ca2+, there was a significant decrease in the radioactivity of the fatty acid of phosphatidylinositol and phosphatidylcholine. Exogenously added arachidonic acid, but not other fatty acids, stimulated the efflux of gamma-aminobutyric acid in the absence of Ca2+. These observations suggest that the release of arachidonic acid from phospholipids is an intrinsic part of the biochemical mechanism that modulates the gamma-aminobutyric acid efflux.     

Cellular interactions between n-6 and n-3 fatty acids: a mass analysis of fatty acid elongation/desaturation, distribution among complex lipids, and conversion to eicosanoids.

The biologic effect of eicosanoids depends in large measure upon the relative masses in tissues of eicosanoids derived from the n-6 fatty acids, dihomogammalinolenic acid and arachidonic acid, and the n-3 fatty acid, eicosapentaenoic acid. Generation of this tissue balance is related to the relative cellular masses of these precursor fatty acids, the competition between them for entry into and release from cellular phospholipids, and their competition for the enzymes that catalyze their conversion to eicosanoids. In order to better understand these processes, we studied the cellular interactions of n-6 and n-3 fatty acids using an essential fatty acid-deficient, PGE-producing, mouse fibrosarcoma cell line, EFD-1. Unlike studies using cells with endogenous pools of n-6 and n-3 fatty acids, the use of EFD-1 cells enabled us to examine the metabolic fate of each family of fatty acids both in the presence and in the absence of the second family of fatty acids. Thus, the specific effects of one fatty acid family on the other could be directly assessed. In addition, we were able to replete the cells with dihomogammalinolenic acid (DHLA), arachidonic acid (AA), and eicosapentaenoic acid (EPA) of known specific activities; thus the masses of cellular DHLA, AA, and EPA, and their metabolites, PGE1, PGE2, and PGE3, respectively, could be accurately quantitated. The major findings of this study were: 1) n-6 fatty acids markedly stimulated the elongation of EPA to 22:5 whereas n-3 fatty acids inhibited the delta 5 desaturation of DHLA to AA and the elongation of AA to 22:4; 2) n-6 fatty acids caused a specific redistribution of cellular EPA from phospholipid to triacylglycerol; 3) n-3 fatty acids reduced the mass of DHLA and AA only in phosphatidylinositol whereas n-6 fatty acids reduced the mass of EPA to a similar extent in all cellular phospholipids; and 4) n-3 fatty acids caused an identical (33%) reduction in the bradykinin-induced release of PGE1 and PGE2, whereas n-6 fatty acids stimulated PGE3 release 2.3-fold. Together, these highly quantitative metabolic data increase our understanding of the regulation of both the cellular levels of DHLA, AA, and EPA, and their availability for eicosanoid synthesis. In addition, these findings provide a context for the effective use of these fatty acids in dietary therapies directed at modulation of eicosanoid production.     

Carnitine inhibits arachidonic acid turnover,platelet function,and oxidative stress

Carnitine is a physiological cellular constituent that favors intracellular fatty acid transport, whose role on platelet function and O(2) free radicals has not been fully investigated. The aim of this study was to seek whether carnitine interferes with arachidonic acid metabolism and platelet function. Carnitine (10-50 microM) was able to dose dependently inhibit arachidonic acid incorporation into platelet phospholipids and agonist-induced arachidonic acid release. Incubation of platelets with carnitine dose dependently inhibited collagen-induced platelet aggregation, thromboxane A(2) formation, and Ca(2+) mobilization, without affecting phospholipase A(2) activation. Furthermore, carnitine inhibited platelet superoxide anion (O(2)(-)) formation elicited by arachidonic acid and collagen. To explore the underlying mechanism, arachidonic acid-stimulated platelets were incubated with NADPH. This study showed an enhanced platelet O(2)(-) formation, suggesting a role for NADPH oxidase in arachidonic acid-mediated platelet O(2)(-) production. Incubation of platelets with carnitine significantly reduced arachidonic acid-mediated NADPH oxidase activation. Moreover, the activation of protein kinase C was inhibited by 50 microM carnitine. This study shows that carnitine inhibits arachidonic acid accumulation into platelet phospholipids and in turn platelet function and arachidonic acid release elicited by platelet agonists.     

Selective inhibition of free arachidonic acid production in activated alveolar macrophages by calmodulin antagonists

The effect of calmodulin antagonists on the amounts of free fatty acids produced by rabbit alveolar macrophages was determined by fluorometric high-performance liquid chromatography. Opsonized zymosan-induced arachidonic acid production was dramatically suppressed in the presence of W-7 and trifluoperazine without an effect on the production of other fatty acids. Calmodulin antagonists inhibited phospholipase A and abolished the release of arachidonic acid from phospholipids. The present results suggest that a zymosan-sensitive pool of 20:4, which is different from that of other fatty acids, is present in macrophages and that calmodulin antagonists selectively inhibit phospholipase A, which preferentially degrades phospholipids with 20:4.     

Ethanol induces release of arachidonic acid but not synthesis of eicosanoids in mouse peritoneal macrophages

Exposure of mouse peritoneal macrophages to ethanol induces a rapid release of arachidonic acid to the extracellular medium. All major classes of phospholipids, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol contribute to this release. Ethanol-induced mobilization of arachidonic acid occurs by deacylation, but it is not accompanied by eicosanoid synthesis. These data suggest that at least two signals are necessary for the release and metabolism of arachidonic acid. Ethanol also activates a phospholipase C which hydrolyzes only phosphatidylinositol, and not its phosphorylated derivatives.     

The appearance of phospholipase and cyclo-oxygenase activities in the human promyelocytic leukemia cell line HL60 during dimethyl sulfoxide-induced differentiation

The human promyelocytic leukemia cell line HL60 can be induced to differentiate into mature granulocytes by exposure to dimethyl sulfoxide. During differentiation a phospholipase activity, which releases arachidonic acid from membrane phospholipids, is expressed. Similarly, fatty acid cyclo-oxygenase activity increases 10-fold. In addition, there is a 40-fold increase in chemotactic formyl peptide receptor binding and a dramatic increase in glucose oxidation via the hexosemonophosphate shunt. The addition of indomethacin, a potent cyclo-oxygenase inhibitor, to the culture medium reduced the cyclo-oxygenase activity of HL60 cells exposed to dimethyl sulfoxide by 97%. However, the presence of indomethacin did not block the dimethyl sulfoxide induced increases in chemotactic formyl peptide receptor binding and hexosemonophosphate shunt activity.     

Reduced labeling of brain phosphatidylinositol,triacylglycerols, and diacylglycerols by [1-14C]arachidonic acid after electroconvulsive shock: Potentiation of the effect by adrenergic drugs and comparison with palmitic acid labeling

The effect of electroconvulsive shock on the labeling of phospholipids and neutral lipids in mice brains was examined after intracerebral injection of [1-¹⁴C] arachidonic acid or [1-¹⁴C]palmitic acid. Electroconvulsive shock reduced greatly the removal of radiolabeled arachidonic acid from the free fatty acid pool. At the same time, the incorporation of arachidonic acid was partially inhibited in triacylglycerol, diacylglycerol, and phosphatidylinositol, whereas the incorporation of [1-¹⁴C]palmitic acid was not affected. Pretreatment with desipramine and pargyline potentiated the lipid effect of electroconvulsive shock in neutral glycerides. These electroconvulsive shock-induced changes reflect alterations in the metabolism of intracerebrally injected arachidonic acid, but not of similarly injected palmitic acid. From the available data whether decreased ATP, enzyme inhibition or other factors are involved cannot be ascertained. Moreover, the electroconvulsive shock-enhanced endogenous free arachidonic acid may possibly dilute the injected radiolabeled fatty acid, thus decreasing its availability for arachidonoyl-coenzyme A synthesis. Hence, a partial inhibition of the activation-acylation of these fatty acids, primarily arachidonic acid, also may be involved in the seizure-induced accumulation of free fatty acids in the brain.     

12-L-hydroxy-5,8,10,14-eicosatetraenoic acid, a chemotactic fatty acid, is incorporated into neutrophil phospholipids and triglyceride

Platelets contain a lipoxygenase which converts arachidonic acid to 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) which has been shown to be chemotactic for human neutrophils and eosinophils. [¹⁴C]-12-HETE was biosynthesized, purified and incubated at a concentration of 1 μM with human neutrophils. Lipids were extracted from the neutrophils and the media, and the radiolabeled products identified. 26 percent of the radiolabel was found in the cells after 30 min incubation, essentially all of it esterified into phospholipid and triglyceride. The radiolabeled phospholipids and triglycerides were transesterified and the liberated fatty acid was identified as [¹⁴C]-12-HETE.This is the first demonstration of direct alteration of membrane components by a chemotactic agent and may be an example of a more generalized mechanism for altering membrane characteristics.     

Chemotactic peptide stimulation of arachidonic acid release in HL60 cells, an interaction between G protein and phospholipase C mediated signal transduction

The mechanism of phospholipase A2 activation by chemotactic peptide was investigated in human promyelocytic HL60 cells. N-Formyl-methionyl-leucyl-phenylalanine (fMetLeuPhe) and the non-hydrolyzable GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]) induced arachidonic acid release in permeabilized and metabolically inhibited HL60 cells, a preparation in which calcium was buffered and inositol phospholipid hydrolysis was inhibited. Inositol phosphate generation and arachidonic acid were shown to be temporally dissociated. These results suggest that receptor-dependent phospholipase C activity is not required for fMetLeuPhe to induce arachidonic acid release. However, fMetLeuPhe effects were highly calcium-dependent and inhibition of phospholipase C reduced fMetLeuPhe stimulation of arachidonic acid release even in the permeabilized cell preparation. We conclude that although phospholipase A2 activation is linked to the fMetLeuPhe receptor independent of phospholipase C, actions of phospholipase C to mobilize calcium and release diacylglycerol may be important to phospholipase A2 activation in the intact cell.     

Effects of postdecapitation ischemia on the metabolism of [14C]arachidonic acid and [14C]palmitic acid in the mouse brain

The effect of postdecapitation ischemia on the labeling of the free fatty acid pool and their incorporation in lipids was examined during the first 10 min after decapitation in mouse brain that had been injected intracerebrally with either [1-14C]arachidonic acid or [1-14C]palmitic acid. One min after decapitation, animals injected with labeled arachidonic acid exhibited a greatly reduced incorporation of label in brain phospholipids, diglycerides, and triglycerides. When radioactive palmitic acid was used, brain lipids exhibited considerably less inhibition of label. However, a similar degree of inhibition was observed 10 min after decapitation with both fatty acids. At this time, free arachidonic acid had decreased 84% as compared to the 24% decrease observed in the controls, and about 77% of the free palmitic acid remained in the free fatty acid fraction as compared with 30% in the controls. This decreased labeling may reflect ATP shortage that affects the fatty acid activation-reacylation reactions or the enzymes involved. Alternatively, the enhanced endogenous free arachidonic acid may compete with the radiolabeled arachidonic acid resulting in an inhibition of lipid labeling. Inhibition of label may have been greater in radiolabeled arachidonic acid than palmitic because of the larger accumulation of the former endogenous fatty acid during early ischemia.     

Prostaglandins may play a signal-coupling role during phagocytosis in Amoeba proteus

Summary Phagocytosis in Amoeba proteus can be induced with prostaglandins (PG). In addition, arachidonic acid (the fatty acid precursor to the PG-2 series) also induces phagocytosis. The induction of phagocytosis with arachidonic acid can be partially inhibited by the cyclooxygenase inhibitor indomethacin. Phagocytosis in the amoeba can also be induced with the chemotactic peptide N-formylmethionyl-leucylphenylalanine (NFMLP). The peptide presumably induces phagocytosis by interacting with receptors on the amoeba surface, which may initiate the release of arachidonic acid from membrane lipids. NFMLP-induced phagocytosis can also be partially inhibited by indomethacin. It is suggested that PG's or biochemically related substances may play a signal-coupling role during phagocytosis in the amoeba.     

Uptake, release and novel species-dependent oxygenation of arachidonic acid in human and animal airway epithelial cells

To determine identities of mediators and mechanisms for their release from pulmonary airway epithelial cells, we examined the capacities of epithelial cells from human, dog and sheep airways to incorporate, release and oxygenate arachidonic acid. Purified cell suspensions were incubated with radiolabeled arachidonic acid and/or ionophore A23187; fatty acid esterification and hydrolysis were traced chromatographically, and oxygenated metabolites were identified using high-pressure liquid chromatography and mass-spectrometry. In each species, cellular uptake of 10 nM arachidonic acid was concentrated in the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine fractions, and subsequent incubation with 5 microM A23187 caused release of 10-12% of the radiolabeled pool selectively from phosphatidylcholine and phosphatidylinositol. By contrast, the products of arachidonic acid oxygenation were species-dependent and in the case of human cells were also novel: A23187-stimulated human epithelial cells converted arachidonic acid predominantly to 15-hydroxyeicosatetraenoic acid (15-HETE) and two distinct 8,15-diols in addition to prostaglandin (PG) E2 and PGF2 alpha. Cell incubation with exogenous arachidonic acid (2.0-300 microM) led to progressively larger amounts of 15-HETE and the dihydroxy, epoxyhydroxy and keto acids characteristic of arachidonate 15-lipoxygenase. Both dog and sheep cells converted exogenous or endogenous arachidonic acid to low levels of 5-lipoxygenase products, including leukotriene B4 without significant 15-lipoxygenase activity. In the cyclooxygenase series, sheep cells selectively released PGE2, while dog cells generated predominantly PGD2. The findings demonstrate that stereotyped esterification and phospholipase activities are expressed at uniform levels among airway epithelial cells from these species, but pathways for oxygenating arachidonic acid allow mediator diversity depending greatly on species and little on arachidonic acid presentation.     

Potentiation of arachidonic acid release by phorbol myristate acetate in platelets is not due to inhibition of arachidonic acid uptake or incorporation into phospholipids

Activators of protein kinase C, such as tumor-promoting phorbol esters (e.g., phorbol myristate acetate), mezerein, (-)-indolactam V and 1-oleoyl 2-acetoyl glycerol, potentiate arachidonic acid release caused by elevation of intracellular Ca2+ with ionophores. This action of protein kinase C-activators required protein phosphorylation, and was attributed to enhanced hydrolysis of phospholipids by phospholipase A2 (Halenda, et al. (1989) Biochemistry 28, 7356-7363). Recently Fuse et al. ((1989) J. Biol. Chem 264, 3890-3895) reported that the apparent enhanced release of arachidonate was actually due to inhibition of the processes of re-uptake and re-esterification of released arachidonic acid. They attributed this to loss of arachidonyl-CoA synthetase and arachidonyl-CoA lysophosphatide acyltransferase activities, which were measured in membranes obtained from phorbol myristate acetate-treated platelets. In this paper, we show that phorbol myristate acetate, at concentrations that strongly potentiate arachidonic acid release, does not inhibit either arachidonic acid uptake into platelets or its incorporation into specific phospholipids. Furthermore, the fatty acid 8,11,14-eicosatrienoic acid, a competitive substrate for arachidonyl-CoA synthetase, totally blocks arachidonic acid uptake into platelets, but, unlike phorbol myristate acetate, does not potentiate arachidonic acid release by Ca2+ ionophores. We conclude that the action of phorbol myristate acetate is to promote the process of arachidonic acid release by phospholipase A2.     

Arachidonic acid release from diacyl phosphatidylethanolamine by human platelet membranes.

At pH9.5 in the presence of 10 mM-Ca2+, human platelet membranes released 22% (167 of 785 nmol) of arachidonic acid that was esterified to phospholipids. With the use of synthetic choline (dinonadecanoyl) and ethanolamine (diheptadecanoyl) phosphoglycerides as internal reference compounds, 115 nmol of the released arachidonic acid was shown to be derived from endogenous breakdown of the phosphatidylethanolamine fraction. Further, the lysophosphatidylethanolamine that was released along with the arachidonic acid was shown virtually to lack fatty aldehydes and to contain a preponderance of fatty acids that have a preference for esterification at the 1-position of naturally occurring phosphatidylethanolamine of human platelets. These findings ruled out plasmalogen phosphatidylethanolamine as the source of the released arachidonic acid. We conclude that diacyl phosphatidylethanolamine was the principal source of arachidonic acid released by human platelet membranes under the conditions described.