Acyltransferase-catalyzed transfer of arachidonic acid to lysophospholipids in rat pancreatic acini

The conversion of 2-lysophospholipids into corresponding phospholipids via acyl-CoA acyltransferase was demonstrated in homogenates of rat pancreatic acini. Arachidonic acid was greatly preferred over stearic acid as the acyl donor. Lysophophosphatidylinositol and lysophosphatidylcholine acyltransferases were distributed in subcellular fractions of acinar homogenates with specific activity highest in the fractions known to contain secretory organelles and mitochondria. The distribution of lysophosphatidylinositol acyltransferase paralleled that of a mitochondrial marker (succinate cytochrome C reductase). These findings extend the evidence implicating arachidonate release and reincorporation into phospholipids as a link in the pathway that culminates in pancreatic secretion.     

Evidence that macrophages transfer arachidonic acid and cholesterol to tissues in vivo

Our previous studies have shown that [(14)C]-labelled cholesterol (CHOL) and arachidonic acid (AA) are transferred from macrophages (Mphi) to lymphocytes (LY) when these cells are co-cultured. In this study, we investigated whether these lipids can be transferred from control and thioglycollate-elicited Mphi (THIO-elicited Mphi) to various tissues and organs in vivo. For this purpose, control and THIO-elicited Mphi were pre-treated with [(14)C]-AA and [(3)H]-CHOL and then injected into the jugular vein of adult rats. More than 75% of the radioactivity injected was found in the liver of rats treated with [(14)C]-AA labelled-Mphi either control and THIO-stimulated. The radioactivity of [(3)H]-CHOL labelled Mphi was transferred mainly to the liver (51% in the control Mphi and 23% in the thioglycollate Mphi7) but it was also found in the kidney, lung and spleen. These results support the proposition that the transfer of lipids between cells also occurs in vivo. The full significance of this phenomenon however remains to be elucidated.     

Ultrastructural localization of arachidonic acid in stimulated macrophages

The distribution of 3[H] arachidonic acid incorporated into cultured mouse peritoneal macrophages was assessed upon stimulation of the cells with either the calcium ionophore A23187 or zymosan. After a labeling time of 24 h, cells were stimulated and processed for light and electron microscopic autoradiography. Grains were primarily localized over the plasma membrane and lipid-containing vesicles of both control and stimulated cells. In macrophages stimulated with ionophore, a decreased labeling density was evident in both of these cell compartments. Similar alterations in labeling pattern were observed in zymosan treated cells, although a larger decline in grain density occurred from the plasma membrane compartment. Immunocytochemical localization of PGE2, a major eicosanoid product released upon ionophore stimulation, revealed the presence of the prostaglandin in clear vesicular structures, many of which appear to be continuous with the plasma membrane. These results provide morphological evidence that different cellular pools of arachidonic acid may be differentially mobilized for eicosanoid production as a function of the mode of stimulation.     

Transfer of arachidonic acid between phospholipids in rat liver microsomes

Phosphatidylcholine and phosphatidylinositol labelled with radioactive oleic, arachidonic or linoleic acids in the 2-acyl position were prepared. Rat liver microsomes were incubated with either lysophosphatidylcholine or lyso-phosphatidylinositol and the opposite 2-acyl-labelled phospholipid, and were found to catalyse a transfer of fatty acids between the two phospholipids. This was shown to be a direct Co-enzyme A-mediated transfer that does not involve a free fatty acid intermediate (i.e. it is independent of phospholipase A₂ activity). Arachidonoyl transfer took place at about four times the rate of linoleoyl transfer; oleoyl transfer was not detectable. The role of direct arachidonoyl transfer to phosphatidylinositol in the controlled release of arachidonic acid for prostaglandin synthesis is discussed.     

Modulation of lymphocyte proliferation by macrophages and macrophages loaded with arachidonic acid

Arachidonic acid (AA) is incorporated and exported by macrophages. This fatty acid is also transferred from macrophages (Mphi) to lymphocytes (LY) in co-culture. This observation led us to investigate the effect of macrophages pre-loaded with AA on concanavalin A (Con A)-stimulated lymphocyte proliferation. The experiments were performed in co-culture. This condition reproduces the in vivo microenvironment in which the modulation of lymphocyte proliferation is dependent on the interaction with macrophages. Lymphocytes obtained from untreated rats or from intraperitoneally thioglycolate-injected rats (THIO-treated) were co-cultured with macrophages from the same rats. Firstly, macrophages were co-cultured for 48 h with Con A-stimulated lymphocytes in different proportions: 0.5, 1, 2.5, 5, 10, 20 and 30% of 5 x 10(5) lymphocytes per well. At 1% proportion, macrophages caused maximum stimulation of lymphocyte proliferation; a four- to five-fold increase, for cells from both thioglycolate-treated and untreated rats, respectively, whereas at 20% it caused maximum inhibition. In addition, 1 or 20% macrophages were pre-loaded with several AA concentrations during a period of 6 h and co-cultured with lymphocytes. At 180 microM AA and 1% macrophages, lymphocyte proliferation was inhibited (by 25%), whereas at 20% macrophages, proliferation was increased, by 25- and three-fold, respectively, for cells from untreated and THIO-treated rats. AA added directly to the medium reduced lymphocyte proliferation, also being toxic to these cells at 100 microM. No toxic effects of AA were observed on macrophages. Additional evidence suggests that nitric oxide production is involved in the modulation of lymphocyte proliferation by AA-pre-loaded macrophages. These findings support the proposition that AA can directly modulate lymphocyte proliferation and the interaction between macrophages and lymphocytes.     

Glomeruli cooperate with macrophages in converting arachidonic acid to prostaglandins and hydroxyeicosatetraenoic acids

The interaction of proliferating macrophages with the glomerulus may play an important role in certain forms of glomerulonephritis. This interaction could involve metabolites of arachidonic acid (C20:4) such as prostaglandins (PG) and lipoxygenase products. We therefore investigated the conversion of exogenous |³H| C20:4 into hydroxyeicosatetraenoic acids (HETE) and PG by isolated glomeruli and macrophages from rats, alone and in combination. As demonstrated by HPLC, glomeruli converted C20:4 predominantly to lipoxygenase products -mainly 12-HETE- and, to a lesser extent, to PG. Resident macrophages converted C20:4 to equivalent amounts of HETE and PG, mainly 12-HETE and 6 keto-PGF_1α. When macrophages and glomeruli were studied in combination, a striking interaction was detected in both pathways of C20:4 metabolism. Production of 6 keto-PGF_1α was stimulated and considerable amounts of TXB₂, PGD₂ and hydroxyheptadecatrienoic acid (HHT) were also produced Total 12-HETE production was unchanged. When a lipid extract of glomeruli, containing oxygenated metabolites of C20:4, was added to macrophages, stimulation of 12-HETE occurred without any change in HHT or PG formation. When, on the contrary, a lipid extract from macrophages was added to glomeruli, 12-HETE production by the glomeruli was nearly completely abolished. Thus the unchanged total 12-HETE production by coincubated glomeruli and macrophages resulted from its increased production by macrophages and its decreased production by glomeruli. These data suggest that interaction between glomeruli and macrophages results in activation of C20:4 metabolism by macrophages followed by inhibition of C20:4 metabolism by glomeruli. Such a regulatory process could play a role in the inflammatory response to immunological injuries during macrophage-dependent human and experimental glomerulonephritis.     

Monoclonal antibodies to CD45 modify LPS-induced arachidonic acid metabolism in macrophages

Signaling by lipopolysaccharide (LPS) through CD14 involves the activation of protein tyrosine kinases of the src family and leads to cytokine production and activation of arachidonic acid metabolism in macrophages. CD45 protein tyrosine phosphatase (PTPase) might play a role in modulating the response through this pathway. Although a critical role in regulation of T-cell signaling for CD45 has been demonstrated, little is known about its role in macrophages. Monoclonal antibodies to CD45 and F(ab')(2) fragments of the monoclonal antibody enhanced the response of differentiated THP-1 monocytic cells to LPS for the release of radiolabeled arachidonic acid metabolites, prostaglandin E(2), and tumor necrosis factor alpha. The enhancing effect of anti-CD45 mAbs was shown to occur primarily through CD14-dependent signaling by performing the experiments under conditions favoring that pathway. Further, LPS may be able to alter the enzymatic activity of CD45, as shown by Western blots of CD45 immunoprecipitates in which LPS caused a transient change in the phosphorylation state of CD45. We conclude that CD45 appears to play a role in LPS-induced responses through the CD14 pathway, possibly through its PTPase activity.     

Fatty acid composition and incorporation of arachidonic acid into phospholipids of hemocytes from the tobacco hornworm Manduca sexta

The fatty acid compositions were determined for total lipids, triacylglycerols, phospholipids and four phospholipid fractions, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine/phosphatidylinositol (PS/PI) and cardiolipin (CA) obtained from hemocytes and cell-free serum from second day, fifth instar larvae of the tobacco hornworm Manduca sexta and the standard Manduca rearing medium. The hemocyte fatty acid profiles were considerably different from the profiles of the medium the insects were reared on and from the profiles of the cell-free serum. Hemocyte neutral lipids had lower proportions of polyunsaturated fatty acids than phospholipids. The fatty acid profiles of PC, PE, PS/PI and CA differ from each other and from the total lipid profiles, indicating selective fatty acid incorporation into hemocyte phospholipid species. Studies with radioactive arachidonic acid similarly indicated selective incorporation of polyunsaturated fatty acids into hemocyte lipids. Under our in vitro conditions, >40% of the total radioactivity was incorporated into hemocyte lipids. About 93% of the incorporated radioactivity was found in phospholipids. Within phospholipids. most of the radioactivity was associated with PC (46%), and less with PE (28%) and PS/PI (21%). Very little radioactivity was recovered in CA (0.9%).     

Cyclooxygenase products of metabolism of arachidonic acid in mouse macrophages exposed to N-phenyllinoleamide from toxic oil samples

N-phenyllinoleamide (NPLA), one of the major extraneous constituents of Spanish toxic oil samples, appears to enhance the cyclooxygenase metabolic pathway of arachidonic acid by peritoneal mouse macrophages. Results reported herein show an increased biosynthesis of 6-oxo-PGF1 alpha and TXB2 by macrophages exposed to NPLA. However, light and electron microscopy failed to show cellular alterations in macrophages incubated with NPLA for two hours at 27 degrees C. These data suggest a possible involvement of cyclooxygenase arachidonic acid metabolism in the etiopathogenesis of the Spanish Toxic Oil Syndrome.     

Feedback regulation of platelet function by 12S-hydroxyeicosatetraenoic acid: inhibition of arachidonic acid liberation from phospholipids

We have proposed a mechanism that platelet aggregation is regulated by its 12-lipoxygenase product, 12S-hydroxyeicosatetraenoic acid (12-HETE) (Sekiya, F., Takagi, J. and Saito, Y. (1989) Thrombos. Res. 56, 407-415). Inhibition of endogenous 12-HETE production by 15-HETE, a specific inhibitor of 12-lipoxygenase, accelerated aggregation of bovine platelets in response to collagen and arachidonic acid liberation from phospholipids was enhanced. Exogenously added 12-HETE suppressed collagen-induced liberation of arachidonic acid and the aggregation was also inhibited. On the other hand, 12-HETE did not interfere with thromboxane synthesis from free arachidonic acid in a cell-free system. These observations suggest that 12-HETE exerts a negative feedback to prevent excess aggregation through interference with arachidonic acid liberation from membrane phospholipids.     

Ionophore-induced metabolism of phospholipids and eicosanoid production in porcine aortic endothelial cells: selective release of arachidonic acid from diacyl and ether phospholipids

Confluent cultures of porcine aortic endothelial cells were prelabeled with 1 microM [14C]arachidonic acid complexed to 1 microM bovine serum albumin. After washing, the cells were stimulated with 1 microM A23187 for time intervals between 30 s and 30 min. Cellular lipids were extracted and separated into major lipid classes and phospholipid subclasses. The external medium was analyzed for released radioactive eicosanoids. The time-course of total release of 14C radioactivity demonstrated a biphasic nature of A23187-induced changes in endothelial cell lipids. Early, from 30 s to 5 min, substantial losses of [14C]arachidonic acid from diacylphosphatidylethanolamine and phosphatidylinositol, as well as an abrupt increase in diacylphosphatidylcholine-associated radioactivity were observed. These initial changes coincided with the release of 14C-labeled cyclooxygenase products. Later changes (5-30 min) included a sustained progressive loss of 14C radioactivity from alkenyl (alk-1-enyl) acylphosphatidylethanolamine and diacylphosphatidylcholine. These later changes coincided with the elaboration of 14C-labeled lipoxygenase products. Although unequivocal assignments cannot be made, the data suggest that specific pools of arachidonic acid provide precursors for individual classes of eicosanoids.     

Hydrocortisone inhibits prostaglandin production but not arachidonic acid release from cultured macrophages

We have investigated the action of hydrocortisosone on arachidonic acid mobilisation in cultures of mouse peritoneal macrophages, mouse L929 cells and the mouse macrophage-like cell line RAW264. Hydrocortisone inhibits both arachidonic acid release and prostaglandin production by L929 cells. However, prostaglandin production by macrophages or RAW264 cells is inhibited with a concomitant stimulation rather than inhibition of arachidonic acid release. These data suggest that hydrocortisone acts at the level of phospholipase activity in fibroblasts but at a later stage of prostanoid production in macrophages.     

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.     

Phosphatidylinositol 3-kinase in zymosan- and bacteria-induced signalling to mobilisation of arachidonic acid in macrophages

Stimulation of mouse peritoneal macrophages with zymosan or bacteria results in activation of 85-kDa cytosolic phospholipase A(2) (cPLA(2)) and release of arachidonate. We have investigated the role of phosphatidylinositol 3-kinase (PtdIns 3-kinase) in the signalling leading to activation of cPLA(2) and release of arachidonate in response to zymosan and the bacterium Prevotella intermedia. The specific PtdIns 3-kinase inhibitor wortmannin completely inhibited zymosan- and bacteria-induced release of arachidonate with an IC(50) value of 10-20 nM. Wortmannin also completely inhibited the zymosan-induced activation of cPLA(2), while the cPLA(2) activation by bacteria was partially inhibited by about 50%. Further experiments showed that zymosan-induced activation of extracellular signal-regulated kinase was inhibited, and bacteria-induced activation of the kinase strongly reduced, in the presence of wortmannin. Also zymosan-induced activation of p38 mitogen-activated protein kinase was inhibited by wortmannin, while p38 activation induced by bacteria was not. The zymosan- and bacteria-induced activation of phospholipase C, as determined by the generation of inositol phosphates, was also inhibited by wortmannin. Moreover, zymosan caused activation of PtdIns 3-kinase, which was totally inhibited by wortmannin. In contrast to zymosan and bacteria, arachidonate release induced by calcium ionophore alone, or further amplified by phorbol ester, was not sensitive to wortmannin. These results suggest that PtdIns 3-kinase constitutes a critical component in the zymosan- and bacteria-induced signalling leading to release of arachidonate and that PtdIns 3-kinase is positioned upstream of phospholipase C in this pathway.     

Metabolism of exogenous arachidonic acid by mouse peritoneal macrophages

On incubation of resident mouse peritoneal macrophages with arachidonic acid several hydroxyacyl derivatives detectable in cellular supernatants are formed. As main products monohydroxyarachidonic acids (monoHETE's) were identified. In addition, smaller amounts of dihydroxyarachidonic acids (diHETE's) supernatants by reversed phase HPLC, normal phase HPLC in combination with UV-spectroscopy and combined gas-chromatography / masspectrometry revealed the presence of 5-, 8-, 12- and 15 - mono-HETE's, two distinct 5, 12-diHETE's, several 8, 15-diHETE's and 14, 15-diHETE. Among the 5, 12-diHETE's, only small amounts of a compound with the characteristics of LTB, were detected. Under the conditions employed, the cycloxygenase products PGE₂ and PGI₂ (as 6-keto-PGF_1g) were only minor metabolites. In contrast, when macrophage cultures were stimulated with the phagocytic stimulus zymosan, PGI₂, PGE₂ and LTC₄ were found as the major conversion products of arachidonic acid, whereas mono- and diHETE's were not formed in detectable amounts.     

Metabolism of exogenous arachidonic acid by mouse peritoneal macrophages

On incubation of resident mouse peritoneal macrophages with arachidonic acid several hydroxyacyl derivatives detectable in cellular supernatants are formed. As main products monohydroxyarachidonic acids (monoHETE's) were identified. In addition, smaller amounts of dihydroxyarachidonic acids (diHETE's) were formed. A detailed analysis of cell culture supernatants by reversed phase HPLC, normal phase HPLC in combination with UV-spectroscopy and combined gas-chromatography/masspectrometry revealed the presence of 5-, 8-, 12- and 15- monoHETE's, two distinct 5,12-diHETE's, several 8,15-diHETE's and 14,15-diHETE. Among the 5,12-diHETE's, only small amounts of a compound with the characteristics of LTB4 were detected. Under the conditions employed, the cyclooxygenase products PGE2 and PGI2 (as 6-keto-PGF1 alpha) were only minor metabolites. In contrast, when macrophage cultures were stimulated with the phagocytic stimulus zymosan, PGI2, PGE2 and LTC4 were found as the major conversion products of arachidonic acid, whereas mono- and diHETE's were not formed in detectable amounts.     

Nicergoline, an anti-aggregating agent which inhibits release of arachidonic acid from human platelet phospholipids

Nicergoline is an inhibitor of human platelet aggregation induced by many agents. It inhibits platelet prostaglandin production induced by thrombin or collagen but not by arachidonic acid. It abolishes the decrease of platelet phospholipid induced by thrombin. Nicergoline appears as a drug with an antiphospholipase activity of human platelets.     

Turnover of arachidonic acid in the major diacyl and ether phospholipids of human platelets

In this work, the uptake and release of [3H]arachidonic acid by the diacyl and ether species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in human platelets were studied. Uptake of [3H]arachidonic acid into 1,2-diacyl-PC and 1,2-diacyl-PE was much greater than into the ether phospholipids of the same class. In [3H]arachidonoyl-labeled platelets stimulated by thrombin, there was a decrease in total [3H] arachidonoyl-PC. This was accounted for mostly by a decrease in 1-acyl-2-[3H]arachidonoyl-PC while the level of 1-O-alkyl-2-[3H]arachidonoyl-PC (a precursor for platelet-activating factor) increased slightly. However, in ionophore A23187-stimulated platelets, the reduction of total [3H]arachidonoyl-PC was due to a decrease in both 1-acyl-2-[3H]arachidonoyl-PC and 1-O-alkyl-2-[3H] arachidonoyl-PC, suggesting that ionophore should yield more platelet-activating factor than thrombin. In both thrombin- and ionophore-stimulated platelets, there was a net increase in total [3H]arachidonoyl-PE. This consisted of a decrease in 1,2-diacyl-PE, which was essentially complete by 1 min, followed by an increase in 1-O-alk-1'-enyl-2-[3H]arachidonoyl-PE, which was slower and not apparent until 3-5 min after thrombin. During reincubation of labeled platelets with saline, the 1-O-alkyl-2-[3H]arachidonoyl-PC increased by a factor of 2, between 0 and 4 h, with no significant change in the radioactivity of any other phospholipid. Thus, upon stimulation of human platelets, arachidonic is released from both 1,2-diacyl-PC and 1,2-diacyl-PE for metabolism by platelet cyclooxygenase and lipoxygenase, while certain ether pools of PC and PE also collect arachidonic acid.     

Regulation of arachidonic acid metabolism in macrophages by immune and nonimmune interferons

Mouse resident peritoneal M phi release AAA and metabolize it into cyclooxygenase- and lipoxygenase-derived eicosanoids, when triggered in vitro with different stimuli. Pretreatment of M phi with nonimmune IFN-alpha and IFN-beta dramatically decreased AA liberation from M phi phospholipids and eicosanoid formation after stimulation of M phi with Zy, A23187, or PMA. M phi exposed to immune IFN-gamma also showed a substantial impairment of both AA liberation and eicosanoid production upon exposure to Zy. However, AA and eicosanoid release was increased by IFN-gamma, rather than depressed, in PMA-triggered M phi. In addition, IFN-gamma showed differential effects on M phi stimulated with A23187. In fact, it inhibited AA release as well as formation of lipoxygenase-derived LTC4, but it highly increased the release of the cyclooxygenase products PGE2 and 6-keto PGF1 alpha. The ability of IFN-gamma to differentially modulate AA metabolism of M phi, depending on the nature of the triggering agent, sets forth the high specificity of the regulatory capacity of this molecule. This is at variance with the down regulation of AA metabolism that is generally observed with nonimmune IFN.