Phospholipid metabolism in human neutrophils activated by N-formyl-methionyl-leucyl-phenylalanine. Degranulation is not required for release of arachidonic acid: studies with neutrophils and neutrophil-derived cytoplasts.

Neutrophils respond to chemoattractants by aggregating, degranulating, remodelling of phospholipids and releasing arachidonic acid. To determine whether ligand-induced remodelling of phospholipids depends on redistribution of intracellular organelles (degranulation), we compared phospholipid remodelling of human neutrophils with that of neutrophil-derived cytoplasts. Cytoplasts, organelle-depleted vesicles of cytosol surrounded by plasmalemma, cannot degranulate. Without a stimulus, [3H]arachidonate was incorporated preferentially into phosphatidylinositol (PI) and phosphatidylcholine (PC). Exposure of cytoplasts and neutrophils prelabelled with [3H]arachidonate or [14C]glycerol to fMet-Leu-Phe (10(-7) M) induced rapid changes in distribution of label and mass of individual phospholipids: [3H]arachidonate in phosphatidic acid (PA) increased 500% (120 s), [14C]glycerol incorporation and mass of PA approached 200% of unstimulated values, and [3H]arachidonate in PI decreased continuously; these data are compatible with activity of a PI/PA cycle. However, the mass of PI in both preparations and [14C]glycerol label in intact neutrophils increased initially (5 s), suggesting net synthesis and mobilization of more than one pool of PI. Heterogeneity of PC pools was also observed: [3H]arachidonate was lost from PC immediately upon addition of stimulus, whereas mass and [14C]glycerol values increased. Thus, net phospholipid synthesis, redistribution of arachidonate and activation of the PI/PA cycle are immediate responses of the neutrophil to receptor occupancy by chemoattractants. Furthermore, the similarity in response to fMet-Leu-Phe of neutrophils and granule-free cytoplasts indicates that these processes are independent of degranulation.     

Inhibition of lectin-induced lymphocyte activation by diamide and other sulfhydryl reagents

Inhibition of lectin-induced lymphocyte activation by five reagents capable of combining with or oxidizing free sulfhydryl groups was examined. Each of the reagents tested was capable of inhibiting [methyl-³H]thymidine or [¹⁴C]uridine incorporation into trichloroacetic acid-insoluble material. Four of these reagents, iodoacetamide and N-ethylmaleimide (alkylating agents) and 5,5′-dithiobis (2-nitrobenzoic acid) and p-hydroxymercuriphenylsulfonic acid (sulfhydryl binding agents), inhibited activation when added to lymphocyte cultures together with lectin or at any time thereafter through 48 hr. In contrast, the sulfhydryl oxidizing agent diazine dicarboxylic acid bis[N,N-dimethylamide] (diamide) was effective only when added within 30–60 min of lectin or when added after 24 hr. This inhibition of lymphocyte activation was not due to decreased intracellular levels of reduced glutathione or to inhibition of binding of lectin to the lymphocyte. These results suggest that maintenance of free sulfhydryl groups is important during the early induction of lymphocyte activation and suggest that an obligatory step or steps in the activation sequence may involve sulfhydryl interactions.     

Effect of hypoglycemia on the brain free fatty acid level and the uptake of fatty acids by phospholipids

The effect of hypoglycemia on the uptake of [1-¹⁴C]arachidonate and [1-¹⁴C]oleate into a synaptosomal and microsomal glycerophospholipids was investigated. In the presence of ATP, Mg²⁺ and CoA, rat brain synaptosomes and micorsomes catalyze the transfer of arachidonate and oleatc into glycerophospholipids. Arachidonate was mainly incorporated into phosphatidylinositol (PI) and phosphatidylcholine (PC), whereas oleate was incorporated into phosphatidylcholine and phosphatidylethanolamine (PE).Hypoglycemia was produced by intraperitoneal injection of 10 or 100 units of crystalline insulin per kg body weight. Two hours after injection the blood glucose level decreased to 10–20 mg%. The content of brain phospholipids was slightly decreased but the change was not statistically significant. The level of free fatty acids (FFA) was increased. More pronounced and reproducible changes were found when hypoglycemia was produced by injection of 100 units of insulin per/kg body weight. Changes in brain cortex were similar to those observed in microsomes and synaptosomes. Hypoglycemia affected the incorporation of arachidonic acid into glycerophospholipids of brain membranes. Uptake of [1-¹⁴C]arachidonate was decreased selectively by 50% (into phosphatidic acid /PA/) when hypogiycemia was produced by injection of 10 units of insulin per kg body weight. The Higher dose of insulin 100 units per kg body weight produced a 20% inhibition of arachidonate incorporation into synaptosomal PI and a 13% decrease of incorporation into microsomal phosphatidylcholine. Incorporation of [1-¹⁴C]oleate into membrane phospholipids was not changed by hypoglycemic insult. It is proposed that the disturbances in fatty acid level, particularly arachidonate, and decreased uptake of arachidonic acid by synaptosomal glycerophospholipids may be responsible for alteration of membrane function and changes of synaptic processes.     

Effect of Hydroperoxy Fatty Acids on Acylation and Deacylation of Arachidonoyl Groups in Synaptic Phospholipids

The effect of hydroperoxy fatty acids on reactions involved in the acylation-deacylation cycle of synaptic phospholipids was studied in vitro, using nerve ending fraction isolated from rat forebrain. 15-Hydroperoxyeicosatetraenoic acid (15-HPETE), 13-hydroperoxylinoleic acid (13-HP 18: 2), and hydroperoxydocosahexaenoic acid (22:6 Hpx), at 25 microM final concentration, all inhibited the incorporation of [1-14C]arachidonate into synaptosomal phosphatidylinositol (PI), phosphatidylcholine (PC), and triacylglycerides by 50-80%. The lowest effective concentration of 15-HPETE and 13-HP 18:2 resulting in significant inhibition of the reacylation of PI was 5 microM, whereas the inhibition of [1-14C]arachidonate incorporation into PC required 10 and 5 microM hydroperoxy fatty acids, respectively. Cumene hydroperoxide and tert-butyl hydroperoxide at concentrations of 100 microM did not inhibit reacylation of PI and PC. Synthesis of labeled arachidonoyl-CoA from [1-14C]arachidonate was decreased by about 50% by 25 microM hydroperoxy fatty acids both in synaptosomes and in the microsomal fraction. Use of [1-14C]arachidonoyl-CoA as a substrate, to bypass the fatty acid activation reaction, revealed that activity of acyltransferase was not affected significantly by 25 microM 15-HPETE and 13-HP 18:2. At the same time, however, the hydrolysis of labeled arachidonoyl-CoA was substantially enhanced. Exposure of synaptosomes to 25 microM fatty acid hydroperoxides did not affect significantly the endogenous concentrations of five major free fatty acids. It is concluded that (1) among synaptic phospholipids, reacylation of PI and PC is the most susceptible to the inhibitory action of fatty acid hydroperoxides, and (2) the enzymes affected by these compounds in nerve endings are arachidonoyl-CoA synthetase and hydrolase.     

Lipoxygenase products of arachidonic acid modulate biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human neutrophils via phospholipase A2

When human neutrophils, previously labeled in their phospholipids with [14C]arachidonate, were stimulated with the Ca2+-ionophore, A23187, plus Ca2+ in the presence of [3H]acetate, these cells released [14C]arachidonate from membrane phospholipids, produced 5-hydroxy-6,8,11,14-[14C]eicosatetraenoic acid (5-HETE) and 14C-labeled 5S,12R-dihydroxy-6-cis,8,10-trans, 14-cis-eicosatetraenoic acid ([14C]leukotriene B4), and incorporated [3H]acetate into platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Ionophore A23187-induced formation of these radiolabeled products was greatly augmented by submicromolar concentrations of exogenous 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), 5-HETE, and leukotriene B4. In the absence of ionophore A23187, these arachidonic acid metabolites were virtually ineffective. Nordihydroguaiaretic acid (NDGA) and several other lipoxygenase/cyclooxygenase inhibitors (butylated hydroxyanisole, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-2-pyrazolidinone) caused parallel inhibition of [14C]arachidonate release and [3H]PAF formation in a dose-dependent manner. Specific cyclooxygenase inhibitors, such as indomethacin and naproxen, did not inhibit but rather slightly augmented the formation of these products. Furthermore, addition of 5-HPETE, 5-HETE, or leukotriene B4 (but not 8-HETE or 15-HETE) to neutrophils caused substantial relief of NDGA inhibition of [3H]PAF formation and [14C]arachidonate release. As opposed to [3H]acetate incorporation into PAF, [3H]lyso-PAF incorporation into PAF by activated neutrophils was little affected by NDGA. In addition, NDGA had no effect on lyso-PAF:acetyl-CoA acetyltransferase as measured in neutrophil homogenate preparations. It is concluded that in activated human neutrophils 5-lipoxygenase products can modulate PAF formation by enhancing the expression of phospholipase A2.     

Selective inhibition of leukotriene C4 synthesis and natural killer activity by ethacrynic acid

We have investigated the role of arachidonic acid (AA) metabolism in natural killer (NK) cell activity. Human nonadherent (NA) peripheral blood lymphocytes were used as effector cells against 51Cr-labeled K562 target cells. Synthesis of leukotriene C4 (LTC4) is dependent on glutathione S-transferase (GST). We have chosen to study three putative GST inhibitors, namely, ethacrynic acid (ET), caffeic acid (CA), and ferulic acid (FA), with regard to NK activity and with regard to their effect on AA metabolism. The GST inhibitors inhibited NK lysis when added directly to the NK assay. The GST inhibitors inhibited LTC4 synthesis as induced by calcium ionophore A23187 in a dose-dependent manner similar to their inhibition of NK activity. However, only ET was selective, for it had little effect on LTB4, 5-hydroxyeicosatetraenoic acid, and prostaglandin E2 synthesis. LTC4 synthesis was associated with the NK-enriched fractions obtained from discontinuous Percoll gradients. NK-specific anti-Leu-11b antibody and C treatment could abrogate NK activity and LTC4 synthesis. ET was also inhibitory when NA cells were cultured at 37 degrees C for 18 hr. In this case, LTC4 could reverse the inhibitory effect of ET. Our data suggest that LTC4 plays an important role in NK activity.     

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.     

Metabolism of [14C]arachidonic acid-labeled lipids in quiescent and OAG-stimulated ascites tumor cells.

1. A rapid uptake and esterification of [14C]arachidonic acid during the first 4 hr of cultivation of ascites cells in serum-deprived medium was observed followed by a fast turnover of the fatty acid. 2. Labeling and turnover of esterified arachidonate in individual phospholipid classes was in the order: phosphatidylcholine (PC) greater than phosphatidylinositol (PI) much greater than phosphatidylinositol-4-phosphate (PIP) and -4,5-bisphosphate (PIP2) greater than phosphatidylethanolamine (PE) greater than PE-plasmalogens. 3. In cells stimulated with 1-oleoyl-2-acetyl-sn-glycerol a transient course of arachidonic acid incorporation into PC, PI, PIP and PIP2 was determined peaking 30 min after stimulation, indicating both esterification and release under these conditions. 4. The release of arachidonate was blocked by quinacrine which is a specific inhibitor of phospholipase A2.     

CD2 triggering stimulates a phospholipase A2 activity beside the phospholipase C pathway in human T lymphocytes

In previous studies we demonstrated the triggering of the phospholipase C (PLC) pathway during the activation of an Ag-specific human CD4+ T lymphocyte clone by a mitogenic pair of CD2 (X11,D66) mAb. Similar conditions were applied to investigate a possible involvement of a phospholipase A2 (PLA2) acting as an additional alternative pathway during human T cell activation. Our results show that arachidonic acid or its derivatives are released after CD2 triggering. This release is largely independent of PLC activation and is mediated by a PLA2 because: 1) phosphatidylcholine is the preferential source of [3H]arachidonate release; 2) [3H]arachidonic acid release and phosphatidylcholine hydrolysis are blocked by two inhibitors of solubilized PLA2, mepacrine, and 4-p-bromophenacylbromide; and 3) we evidenced a PLA2 activity in cell homogenates. Extracellular calcium appears to play a critical role because the effects of CD2 mAb were inhibited in a Ca2(+)-depleted medium. In contrast, protein kinase C is not implicated since PMA, a protein kinase C activator, neither stimulated arachidonic acid release nor modulated CD2-induced arachidonic acid release. Cyclic AMP which has been proved to regulate the activity of the PLC in T lymphocytes does not appear to play an important role in the regulation of PLA2 activity since PGE2 has only a minimal effect on [3H]-arachidonate release. Altogether, these findings suggest that CD2 triggering stimulates a PLA2 activity in T lymphocytes via an extracellular Ca2(+)-dependent PLC protein kinase C independent mechanism.     

In vitro stimulation of natural killer (NK) cells by soluble factor(s) generated in antigen-stimulated rhesus monkey peripheral blood lymphocyte cultures.

Peripheral blood lymphocytes from rhesus monkeys (Macaca mulatta) sensitized to keyhole limpet hemocyanin (KLH), when stimulated in vitro with KLH, developed natural killer (NK) cell activity that was assayed with Rous Sarcoma virus-transformed marmoset fibroblasts as targets in a 4-hr 51Cr-release assay. The supernatant fluids from 24- to 25-hr KLH-activated cultures were capable of stimulating NK development in nonsensitive lymphocyte cultures. The effector cells were neither macrophages nor B cells (plastic and nylon-wool nonadherent) and did not form E-rosettes with neuraminidase-treated sheep red blood cells. Cultures depleted of EA-rosetting cells, i.e., Fc-receptor-bearing lymphocytes, were incapable of generating NK activity when stimulated in vitro. Kinetic studies showed that peak DNA synthesis, as measured by 3H-T incorporation, preceded maximum cytotoxicity. Elimination of dividing cells by 5-bromo-2'deoxyuridine (BrdU) and light treatment during the interval from day 1 to day 4 inhibited the development of cytotoxicity on day 7. Cell replication was required for the induction of NK cells with KLH as well as with antigen-activated culture supernatant fluids. When cultures were left unstimulated for 4 days, NK activity could not be developed subsequently either by adding antigen, mitogen (PHA), or supernatant fluids from activated cultures.     

The effect of hydrocortisone on cholesterol metabolism of cultured human skin fibroblasts

Hydrocortisone in physiologic concentrations resulted in a reduction in sterol synthesis by cultured normal human skin fibroblasts. These changes were observed when [14C]acetate, [14C]octanoic acid and 3H2O were used as precursors. However, the incorporation of [3H]mevalonic acid lactone into digitonin-precipitable sterols was not affected by hydrocortisone, suggesting that hydrocortisone inhibits sterol synthesis at a site prior to the formation of mevalonic acid. In contrast, the activity of hydroxymethylglutaryl-CoA reductase was stimulated several-fold by the hormone. Thus, the inhibitory effect of hydrocortisone on the cholesterol synthetic pathway may be on hydroxymethylglutaryl-CoA synthase.     

Inhibition of arachidonic acid incorporation into erythrocyte phospholipids by peracetic acid and other peroxides. Role of arachidonoyl-CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase

To explore possible mechanisms of the arachidonic acid deficiency of the red blood cell membrane in alcoholics, we compared the effect of ethanol and its oxidized products, acetaldehyde and peracetic acid, with other peroxides on the accumulation of [14C]arachidonate into RBC membrane lipids in vitro. Incubation of erythrocytes with 50 mM ethanol or 3 mM acetaldehyde had no effect on arachidonate incorporation. Pretreatment of erythrocytes with 10 mM hydrogen peroxide, 0.1 mM cumene hydroperoxide or 0.1 mM t-butyl hydroperoxide had little effect on [14C]arachidonate incorporation in the absence of azide. However, pretreatment of cells with N-ethylmaleimide, 0.1 mM peracetic acid or performic acid, with or without azide, inhibited arachidonate incorporation into phospholipids but not neutral lipids. In chase experiments, peracetate also inhibited transfer of arachidonate from neutral lipids to phospholipids. To investigate a possible site of this inhibition of arachidonate transfer into phospholipids by percarboxylic acids, we assayed a repair enzyme, arachidonoyl CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase (EC 2.3.1.23). As in intact cells, phospholipid biosynthesis was inhibited more by N-ethylmalemide and peracetic acid than by hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide. Peracetic acid was the only active inhibitor among ethanol and its oxidized products studied and may deserve further examination in ethanol toxicity.     

Incorporation of 5,8,11,14-eicosatetraynoic acid (ETYA) into cell lipids: Competition with arachidonic acid for esterification

5,8,11,14-eicosatetraynoic acid (ETYA), a widely used inhibitor of cyclooxygenase and lipoxygenase, inhibited the incorporation of ¹⁴C-arachidonic acid into cell lipids of the murine thymoma EL4 whereas oleic acid had no effect. Inhibition appeared to result from the ability of ETYA to compete with arachidonic acid for esterification enzymes and to be itself incorporated into cell lipids. The positional specificity for ETYA incorporation was similar to that of arachidonic acid. ETYA, but not oleic acid competed with arachidonate for activation by a selective arachidonoyl CoA synthetase in lymphocytes. This may explain in part the apparent specificity of effects seen on incorporation into whole cells. In addition ETYA, unlike other arachidonate analogs tested previously, caused significant inhibition of the nonselective acyl CoA synthetase in lymphocytes. These results are discussed with respect to the use of ETYA to examine the role of intrinsic arachidonic acid metabolism in cellular processes.     

Radiolabelling of the monate moiety in the study of pseudomonic acid biosynthesis

Abstract Monic acid A was isolated from a Pseudomonas fluorescens fermentation in which pseudomonic acid A (PA) was the principal secondary metabolite. [3-¹⁴C]3-Hydroxy-3-methyl-glutaric acid (HMG) given early in the idiophase radiolabelled PA (1.1% incorporation), confirming experimentally the putative direct involvement of HMG in the biosynthesis of PA, but contributed relatively insignificant radiolabel to the monic acid extracted from the broth at the end of the fermentation. Ethionine inhibited (80%) PA biosynthesis and correspondingly reduced incorporation of [¹⁴C]HMG. In contrast, ethionine increased incorporation of [methyl-¹⁴C]methionine into PA and enhanced specific radioactivity of the antibiotic 8-fold. Ethionine inhibition of secondary metabolite methylations did not divert pseudomonate biosynthesis to give unusual analogues, implying that methylation of a putative pentaketide precursor of the monate moiety forms a vital intermediate of the pseudomonate pathway, but caused a new [¹⁴C]HMG-derived polar metabolite of biosynthetic interest to become evident.     

Phosphatidylinositol-specific phospholipase-C of platelets: association with 1,2-diacyglycerol-kinase and inhibition by cyclic-AMP.

Horse platelets prelabeled with [¹⁴C]arachidonate (AA) rapidly degrade [¹⁴C]phosphatidylinositol (PI) to [¹⁴C]1,2-diacylglycerol (DG) upon treatment with deoxycholate (DOC). This phospholipase-C (PLC) activity is specific for PI since other phospholipids or neutral lipids are not affected. Although exogenous Ca²⁺ is not required for activity, EGTA or EDTA abolishes PI degradation. Addition of Mg²⁺ (1 mM) and ATP (1 mM) results in phosphorylation of the DG and production of phosphatidic acid (PA). Higher concentrations of DOC inhibit DG-kinase. These observations, together with the fact that different platelet agonists induce a rapid degradation of PI and production of PA, indicate that PLC and DG-kinase activities are intimately linked. Incubation of platelets with dibutyryl cyclic-AMP, cyclic AMP-phosphodiesterase inhibitors and pyridoxal-5′-phosphate, which prevent platelet aggregation, inhibits the DOC-dependent conversion of PI to DG. The activity of PLC may play a central role in mediating platelet function and aggregation.     

Plasminogen activator is an apparent lymphocyte mitogen

Culture fluids of avian sarcoma virus (ASV)-transformed but not normal chicken embryo cells frequently elicited a mitogenic response in normal avian and murine lymphocytes. We examined the possibility that plasminogen activator (PA) might be responsible for the observed mitogenic effect. PA activity, present in culture medium, was correlated positively with lymphocyte mitogenic capacity. Treatment of cells with phorbol myristate acetate, which elevates PA levels, increased mitogenesis. Similar treatment with dexamethasone, which inhibits PA biosynthesis and/or secretion, reduced lymphocyte mitogenic activity. Addition to culture fluids of either benzamidine or diisopropylfluorophosphate, both specific PA inhibitors, blocked lymphocyte proliferative responsiveness to culture fluids. In contrast, neither epsilon-amino-caproic acid nor trasylol, which inhibits plasmin esterase activity but not PA, abrogated lymphocyte responsiveness. Furthermore, purified urokinase, an enzyme of similar substrate specificity to PA, had lymphocyte stimulatory activity. These results strongly suggest that PA can function as a lymphocyte mitogen.     

Stimulation of prostaglandin synthesis in WI-38 human lung fibroblasts following inhibition of phospholipid acylation by p-hydroxymercuribenzoate

The release of arachidonic acid and its metabolites, prostaglandin E2 and thromboxane A2, from WI-38 human lung fibroblasts was modulated by p-hydroxymercuribenzoate. Exposure to the inhibitor resulted in a dose-dependent decrease in [1-14C]arachidonic acid uptake and incorporation into phospholipids and neutral lipid pools. Activities of lung fibroblast arachidonyl-CoA synthetase and lysolecithin acyltransferase were inhibited by 100 microM p-hydroxymercuribenzoate. [14C]Arachidonic acid labelled fibroblasts exhibited an increased release of [14C]arachidonate and [14C]prostaglandin E2 of 54% and 112%, respectively, when exposed to 100 microM of inhibitor. The stimulatory effects of 8.0 microM delta 1-tetrahydrocannabinol on arachidonate release and prostaglandin E synthesis (Burstein, S., Hunter, S.A., Sedor, C. and Shulman, S. (1982) Biochem. Pharmacol. 31, 2361-2365) were modified by the inclusion of inhibiting agent, resulting in a 608% stimulation in arachidonic acid release, while prostaglandin E2 and thromboxane A2 synthesis increased 894% and 390%, respectively, over levels obtained by untreated cells. The levels of arachidonate metabolites were altered by inhibitor when compared to cells treated with cannabinoid alone. No significant inhibition by delta 1-tetrahydrocannabinol was found on arachidonic uptake in these cells. In unlabelled studies, p-hydroxymercuribenzoate resulted in a profound, dose-dependent stimulation of prostaglandin E synthesis of 1490% at 150 microM inhibitor concentration. These results provide evidence that free arachidonate is reincorporated via acylation, thereby implicating this pathway as a possible control mechanism for the synthesis of arachidonic acid metabolites.     

Mechanism of arachidonic acid release in human polymorphonuclear leukocytes

Previous studies have demonstrated that [³H]arachidonic acid is released from prelabeled human neutrophil phospholipids when the cells are stimulated by calcium ionophore A23187 or by opsonized zymosan. Neither lysophospholipid generated by phospholipase A₂ activity, diacylglycerol nor monoacylglycerol produced via phospholipase C/diacylglycerol lipase action have been identified following neutrophil challenge. The inability to detect any intermediates during the release of arachidonate is due to either rapid reacylation of lysophospholipid or conversion of diacylglycerol (monoacylglycerol) to cellular acylglycerols. The addition of exogenous [¹⁴C]fatty acid at the time of challenge was employed to determine the involvement of either phospholipase A₂ or phospholipase C activities. Neutrophil stimulation with calcium ionophore A23187 resulted in an incorporation of exogenous [¹⁴C]arachidonate into phosphatidylinositol and phosphatidylcholine, those phospholipids which specifically release arachidonate. When the saturated fatty acid, [¹⁴C]stearate, replaced [¹⁴C]arachidonate, very little [¹⁴C]fatty acid was incorporated into any of the phospholipid species. Lipid phosphorus measurements revealed no significant mass change in any phospholipid class following ionophore challenge. Production of [¹⁴C]phosphatidic acid was not detected, as would be expected if diacylglycerol kinase and de novo phospholipid metabolism were significantly involved.     

Cholesterol inhibits glutamine metabolism in LLC WRC256 tumour cells but does not affect it in lymphocytes: possible implications for tumour cell proliferation

The effect of cholesterol on proliferation and glutamine metabolism of lymphocytes and tumour cells was investigated. The addition of cholesterol to the culture medium did not cause a significant effect on [2-(14)C]-thymidine incorporation in lymphocytes. In the presence of concanavalin A, lymphocyte proliferation was increased by cholesterol (from 0.013 up to 1.3 microm). At high concentrations (234 and 468 microm), however, a marked inhibition of lymphocyte proliferation occurred. The same inhibitory effect was observed in the presence of lipopolysaccharides. Cholesterol also caused a marked decrease of LLC WRC256 tumour cell growth at 117 and 234 microm. The same findings were obtained by the measurement of [2-(14)C]-thymidine incorporation in these cells. The effect of cholesterol on phosphate-dependent glutaminase activity was then tested in cultured lymphocytes and LLC WRC256 tumour cells. Cholesterol at concentrations of 117 and 234 microm did not alter this enzyme activity in lymphocytes. However, this sterol, already at 26 microm, caused a 44 per cent reduction in glutaminase activity. Similar to the changes observed for glutaminase, cholesterol reduced glutamine oxidation in LLC WRC256 tumour cells, whereas no effect was observed on lymphocytes. Therefore, cholesterol might control lymphocyte and tumour cells proliferation by different mechanisms. The significance of these findings for the immune function in tumour-bearing patients remains to be investigated.