Metabolism of 15-hydroxyeicosatetraenoic acid by Caco-2 cells

Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated with [1-14C]15-hydroxyeicosatetraenoic acid (15-HETE), a lipid mediator of inflammation, and [1-14C]arachidonic acid. Both fatty acids were taken up readily and metabolized by Caco-2 cells. [1-14C]Arachidonic acid was directly esterified in cellular phospholipids and, to a lesser extent, in triglycerides. When [1-14C]15-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, about 10% was directly esterified into cellular lipids but most (55%) was beta-oxidized to ketone bodies, CO2, and acetate, with very little accumulation of shorter carbon chain products of partial beta-oxidation. The radiolabeled acetate generated from beta-oxidation of [1-14C]15-hydroxyeicosatetraenoic acid was incorporated into the synthesis of new fatty acids, primarily [14C]palmitate, which in turn was esterified into cellular phospholipids, with lesser amounts in triglycerides. Caco-2 cells were also incubated with [5,6,8,9,11,12,14,15-3H]15-hydroxyeicosatetraenoic acid; most of the radiolabel was recovered either in ketone bodies or in [3H]palmitate esterified in phospholipids and triglycerides, demonstrating that most of the [3H]15-hydroxyeicosatetraenoic acid underwent several cycles of beta-oxidation. The binding of both 15-hydroxyeicosatetraenoic acid and arachidonic acid to hepatic fatty acid binding protein, the only fatty acid binding protein in Caco-2 cells, was measured. The Kd (6.0 microM) for 15-HETE was three-fold higher than that for arachidonate (2.1 microM).     

Specific incorporation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid into phosphatidylcholine in human endothelial cells

The incorporation of hydroxyeicosatetraenoic acids (HETEs) into cellular lipids was studied in cultures of human umbilical vein endothelial cells. 5-[3H]HETE was incorporated into the phospholipids (8%) and neutral lipids (15.5%). The uptake was at half maximum after 15 min and reached a plateau after 1 h. The incorporation occurred mainly into phosphatidylcholine (6.3%) with minimal uptake into phosphatidylserine and phosphatidylinositol (0.6%) or phosphatidylethanolamine (1.2%). There was no uptake of 12-[3H]HETE, 15-[3H]HETE or [3H]leukotriene B4 into phospholipids. Treatment of the phosphatidylcholine fraction with phospholipase A2 released 64% of the 5-[3H]HETE with 26% remaining in the lysophosphatidylcholine fraction. This indicates that the majority of the 5-HETE was in the sn-2 position. Unlabeled 5-HETE and arachidonic acid inhibited the uptake of 5-[3H]HETE into phosphatidylcholine with an ID50 of 2.5 and 1.25 microM, respectively. Stearic acid and 15-HETE were not effective inhibitors. Histamine, which activates phospholipases, increased the uptake of 5-[3H]HETE into phosphatidylcholine by 3-fold. Both 5-[3H]HETE and 12-[3H]HETE were incorporated into the neutral lipids of the cells. Analysis of the neutral lipid fraction revealed that 5-[3H]HETE was incorporated into mono-, di- and triacylglycerols but not cholesterol esters. Incorporation of 5-HETE into cellular lipids reduced histamine- and arachidonic acid-stimulated synthesis of 6-ketoprostaglandin F1 alpha and prostaglandin E2 in a concentration-related manner. Angiotensin I converting enzyme activity was not changed. Thus, 5-HETE is incorporated specifically into phosphatidylcholine and glycerol esters of human endothelial cells and this incorporation inhibits prostaglandin synthesis in these cells.     

Localization of 12-hydroxyeicosatetraenoic acid in endothelial cells

Bovine aortic endothelial cells take up 12-hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase product formed from arachidonic acid. The uptake of [3H]12-HETE reached a maximum in 2 to 4 h. At this time, from 75 to 80% of the incorporated radioactivity was contained in phospholipids, about 85% of the esterified radioactivity remained in the form of 12-HETE, and at least 90% of the phospholipid radioactivity was present in the sn-2-position. Subcellular fractionation on Percoll and sucrose gradients demonstrated that 65 to 74% of the radioactivity was present in membranes enriched in NADPH-cytochrome c reductase and UDP-galactosyl transferase. The specific radioactivity relative to protein of these intracellular membranes was 2.9-times higher than in a plasma membrane fraction enriched in 5'-nucleotidase. A similar intracellular localization was observed when [3H]5-HETE or [3H]arachidonic acid were taken up. The 12-HETE was contained primarily in the choline glycerophospholipids of the microsomal membranes. After incorporation, [3H]12-HETE was removed from the cell lipids much more rapidly than [3H]arachidonic acid, and 80% of the radioactivity released into the medium during the first hour remained as 12-HETE. Because it accumulates in microsomal membranes, 12-HETE uptake may perturb certain intracellular processes and thereby lead to endothelial dysfunction. The relatively rapid removal of the newly incorporated 12-HETE may be an important protective mechanism that prevents excessive accumulation and more extensive endothelial damage.     

Incorporation and effect of arachidonic acid on the growth of human myeloma cell lines

The objectives of this work are to investigate the incorporation of arachidonic acid (AA) in the human myeloma cell lines OPM2, U266 and IM9, and to assess the effect of AA and lipoxygenase products of AA on their growth. The kinetics of acylation of [3H]AA indicates that myeloma cells incorporate AA into their membrane phospholipids and triglycerides. PLA2-treatment and base hydrolysis experiments confirm that [3H]AA is incorporated unmodified in U266, IM9 and OPM2 phospholipids, and is linked by an ester bond. Prelabeling-chase experiments indicate no trafficking of labeled AA among the various phospholipid species. Addition of AA and lipoxygenase products of AA (leukotriene B4 and C4, lipoxin A4 and B4, 12- and 15-hydroxyeicosatetraenoic acid) have no effect on U266, IM9 and OPM2 proliferation assessed by [3H]thymidine incorporation into DNA. In conclusion, while human myeloma cells readily incorporate AA in their membrane phospholipids and triglycerides, AA and lipoxygenase products are not important modulators of their proliferation.     

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.     

Activation of leukocyte movement and displacement of [3H]leukotriene B4 from leukocyte membrane preparations by (12R)- and (12S)-hydroxyeicosatetraenoic acid

Both (12R)- and (12S)-hydroxyeicosatetraenoic acid were demonstrated to produce aggregation of rat leukocytes and enhance human leukocyte chemokinesis. (12R)-Hydroxyeicosatetraenoic acid was 10-20-fold more potent than (12S)-hydroxyeicosatetraenoic acid but at least 500-fold less potent than leukotriene B4 in these assays. These relative potencies are correlated with the potencies of (12R)- and (12S)-hydroxyeicosatetraenoic acid for competition of [3H]leukotriene B4 binding to rat and human leukocyte membrane preparations.     

15-Hydroxyeicosatetraenoic acid (15-HETE) receptors. Involvement in the 15-HETE-induced stimulation of the cryptic 5-lipoxygenase in PT-18 mast/basophil cells.

The mechanisms of stimulation of the inactive 5-lipoxygenase in mast/basophil PT-18 cells by microM 15-hydroxyeicosatetraenoic acid (15-HETE) was investigated. Treatment of PT-18 cells with pM 15-[3H]HETE at 4 degrees for 3 h resulted in the cell association of 10% of the ligand: two-thirds was incorporated into cellular lipids and a third was bound to specific 15-HETE cellular binding sites. Binding data analysis indicated a single class of 15-HETE binding sites with a Kd of 162 nM and a Bmax of 7.1 x 10(5) sites/cell. Unlabeled 15-HETE, 12-HETE, and 5,15-diHETE inhibited the binding of 15-[3H]HETE to cells, whereas LTB4 and PGF2 alpha were relatively ineffective. 2.4 microM 15-HETE (unlabeled) prevented 50% 15-[3H]HETE incorporation. Examination of the effects of 15-HETE methyl ester, 12-HETE, 5,15-diHETE, and pertussis toxin on both the 15-HETE-induced 5-lipoxygenase activation and 15-HETE cell association processes indicated a preponderant correlation of this activation process with specific 15-HETE binding rather than 15-HETE incorporation into phospholipids. In addition, 5,15-diHETE itself stimulated the inactive 5-lipoxygenase and eight times more [3H]diHETE was bound to cells than became incorporated into cellular lipids. The results support the involvement of low affinity 15-HETE receptors, rather than 15-HETE incorporation into cellular lipids, in the 15-HETE-induced stimulation of the 5-lipoxygenase in PT-18 cells.     

Phorbol myristate acetate and the calcium ionophore A23187 synergistically induce release of LTB4 by human neutrophils: involvement of protein kinase C activation in regulation of the 5-lipoxygenase pathway

Phorbol myristate acetate (PMA), a tumor-promoting phorbol ester, and the calcium ionophore A23187 synergistically induced the noncytotoxic release of leukotriene B4 (LTB4) and other 5-lipoxygenase products of arachidonic acid metabolism from human neutrophils. Whereas neutrophils incubated with either A23187 (0.4 microM) or PMA (1.6 microM) alone failed to release any 5-lipoxygenase arachidonate products, neutrophils incubated with both stimuli together for 5 min at 37 degrees C released LTB4 as well as 20-COOH-LTB4, 20-OH-LTB4, 5-(S),12-(R)-6-trans-LTB4, 5-(S),12-(S)-6-trans-LTB4, and 5-hydroxyeicosatetraenoic acid, as determined by high pressure liquid chromatography. This synergistic response exhibited concentration dependence on both PMA and A23187. PMA induced 5-lipoxygenase product release at a concentration causing a half-maximal effect of approximately 5 nM in the presence of A23187 (0.4 microM). Competition binding experiments showed that PMA inhibited the specific binding of [3H]phorbol dibutyrate ([3H]PDBu) to intact neutrophils with a 50% inhibitory concentration (IC50) of approximately 8 nM. 1-oleoyl-2-acetyl-glycerol (OAG) also acted synergistically with A23187 to induce the release of 5-lipoxygenase products. 4 alpha-phorbol didecanoate (PDD), an inactive phorbol ester, did not affect the amount of lipoxygenase products released in response to A23187 or compete for specific [3H]PDBu binding. PMA and A23187 acted synergistically to increase arachidonate release from neutrophils prelabeled with [3H]arachidonic acid but did not affect the release of the cyclooxygenase product prostaglandin E2. Both PMA and OAG, but not PDD, induced the redistribution of protein kinase C activity from the cytosol to the membrane fraction of neutrophils, a characteristic of protein kinase C activation. Thus, activation of protein kinase C may play a physiologic role in releasing free arachidonate substrate from membrane phospholipids and/or in modulating 5-lipoxygenase activity in stimulated human neutrophils.     

Phospholipase A2 activation in human neutrophils. Differential actions of diacylglycerols and alkylacylglycerols in priming cells for stimulation by N-formyl-Met-Leu-Phe

Both 1,2-diacyl- and 1-O-alkyl-2-acylglycerols are formed during stimulation of human neutrophils (PMN), and both can prime respiratory burst responses for stimulation by the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP); however, mechanisms of priming are unknown. Arachidonic acid (AA) release through phospholipase A2 activation and metabolism by 5-lipoxygenase are important activities of PMN during inflammation and could be involved in the process of primed stimulation. Therefore, we have examined the ability of diacyl- and alkylacylglycerols to act as priming agents for AA release and metabolism in human neutrophils. After prelabeling PMN phospholipids with [3H]AA, priming was tested by incubating human PMN with the diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), or its alkylacyl analog, 1-O-delta 9-octadecenyl-2-acetylglycerol (EAG) before stimulating with fMLP. fMLP (1 microM), OAG (20 microM), or EAG (20 microM) individually caused little or no release of labeled AA. However, after priming PMN with the same concentrations of either OAG or EAG, stimulation with 1 microM fMLP caused rapid (peak after 1 min) release of 6-8% of [3H]AA from cellular phospholipids; total release was similar with either diglyceride. Priming cells with OAG also enhanced conversion of released AA to leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) upon subsequent fMLP stimulation, but AA metabolites were not increased in EAG-primed PMN. If fMLP was replaced with the calcium ionophore A23187 (which directly causes release of AA and production of LTB4 and 5-HETE), priming by both diglycerides again enhanced release of [3H]AA, but only OAG priming increased lipoxygenase activity. Indeed, EAG pretreatment markedly reduced LTB4 and 5-HETE production. Thus, both diglycerides prime release of AA from membrane phospholipids but have opposite actions on the subsequent metabolism of AA.     

Incorporation of hydroxyeicosatetraenoic acids into cellular lipids of adrenal glomerulosa cells: inhibition of aldosterone release by 5-HETE

Metabolites of arachidonic acid appear to be involved in the regulation of aldosterone secretion. Adrenal cells metabolize arachidonic acid to several products including hydroxyeicosatetraenoic acids (HETEs). Since HETEs may be incorporated into the membrane lipids in some cells, we investigated whether HETEs were incorporated into lipids of adrenal glomerulosa cells and tested the influence of incorporation on aldosterone secretion. Cells were incubated with [3H] -arachidonic acid, -5-HETE, -12-HETE, -15-HETE or -LTB4. The cellular lipids were extracted and analyzed by TLC. Arachidonic acid was incorporated into all of the cell lipids with greatest accumulations in phospholipids (22%), cholesterol esters (50%), and triglycerides (21%). Uptake was maximal by 30 min. 5-HETE was incorporated into diglycerides and monoglycerides but not into phospholipids or other neutral lipids. The uptake followed a similar temporal pattern as arachidonic acid. 12-HETE was incorporated to a small extent into phospholipids, predominantly phosphatidylcholine. Neither 15-HETE or LTB4 were associated with cellular lipids. Angiotensin increased the uptake of 5-HETE and arachidonic acid into phosphatidylinositol/phosphatidylserine without altering uptake into the other lipids. When cells were pretreated with 5-HETE and washed to remove the unesterified HETE, basal aldosterone release as well as release stimulated by angiotensin, potassium and ACTH were significantly reduced. 15-HETE, which is not incorporated into cellular lipids, was without effect on aldosterone secretion. These studies indicate that 5-HETE may be incorporated into the cellular lipids of adrenal cells and may modulate steroidogenesis.     

Beta-oxidation of medium chain (C8-C14) fatty acids studied in isolated liver cells

The beta-oxidation and esterification of medium-chain fatty acids were studied in hepatocytes from fasted, fed and fructose-refed rats. The beta-oxidation of lauric acid (12:0) was less inhibited by fructose refeeding and by (+)-decanoyl-carnitine than the oxidation of oleic acid was, suggesting a peroxisomal beta-oxidation of lauric acid. Little lauric acid was esterified in triacylglycerol fraction, except at high substrate concentrations or in the fructose-refed state. With [1-14C]myristic acid (14:0), [1-14C]lauric acid (12:0), [1-14C]octanoic acid (8:0) and [2-14C]adrenic acid (22:4(n - 6] as substrate for hepatocytes from carbohydrate-refed rats, a large fraction of the 14C-labelled esterified fatty acids consisted of newly synthesized palmitic acid (16:0), stearic acid (18:0) and oleic acid (18:1) while intact [1-14C]oleic acid substrate was esterified directly. With [9,10-3H]myristic acid as the substrate, small amounts of shortened 3H-labelled beta-oxidation intermediates were found. With [U-14C]palmitic acid, no shortened fatty acids were detected. It was concluded that when the mitochondrial fatty acid oxidation is down-regulated such as in the carbohydrate-refed state, medium-chain fatty acids can partly be retailored to long-chain fatty acids by peroxisomal beta-oxidation followed by synthesis of C16 and C16 fatty acids which can then stored as triacylglycerol.     

Stereoselective hydrogen abstraction in leukotriene A4 synthesis by purified 5-lipoxygenase of porcine leukocytes

Arachidonate 5-lipoxygenase purified from porcine leukocytes transformed arachidonic acid to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid. By the leukotriene A synthase activity of the same enzyme the product was further metabolized to leukotriene A4 (actually detected as 6-trans-leukotriene B4, 12-epi-6-trans-leukotriene B4, and 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acids). The enzyme was incubated with [10-DR-3H]- or [10-LS-3H]-labeled arachidonic acid, and 6-trans-LTB4 and its 12-epimer were analyzed. More than 90% of 10-DR-hydrogen was lost while about 100% of 10-LS-hydrogen was retained, indicating a stereospecific hydrogen elimination from C-10 during the formation of leukotriene A4.     

Stimulation of platelet-activating factor synthesis by a nonmetabolizable bioactive analog of platelet-activating factor and influence of arachidonic acid metabolites

Platelet-activating factor (PAF) is a potent neutrophil agonist operating through specific receptors located on the cell surface. Binding of PAF to its receptor may also stimulate further PAF synthesis, thus providing a means of amplifying the PAF signal for the cell of origin and/or other responsive cells. In this report we demonstrate that 1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (C-PAF), a nonmetabolizable bioactive analog of PAF, stimulates human neutrophils to synthesize PAF, as detected by [3H]acetate incorporation into PAF. This approach allowed us to conclude that [3H]acetate-labeled PAF was formed from endogenous precursor rather than mere turnover of the stimulatory dose of PAF. PAF's ability to initiate further PAF synthesis was confirmed by measuring the PAF-stimulated conversion of 1-O-[3H]alkyl-2-acylglycerophosphocholine to 1-O-[3H]alkyl-2-acetylglycerophosphocholine by prelabeled human neutrophils and by determining the molecular species of 1-O-alkyl-2-[3H]acetylglycerophosphocholine produced by cells stimulated with a single molecular species of PAF (C15:0). Degradation of exogenously added [3H]PAF was not inhibited by C-PAF/5-hydroxyeicosatetraenoic acid treatment. Thus, inhibition of PAF degradation was ruled out as the mechanism accounting for the appearance of labeled PAF in the stimulated cells. Synthesis of PAF in response to C-PAF was not dependent on cytochalasin B pretreatment but was dramatically potentiated by 5-hydroxyeicosatetraenoic acid, which alone was without effect. Additionally, we have demonstrated that another major arachidonate metabolite of neutrophils, leukotriene B4, stimulates PAF production. Thus, at least three products of activated neutrophils, including PAF itself, can promote PAF synthesis by these cells. This positive feedback effect may amplify autacoid production and the final cellular response.     

Corticosteroid effect on Caco-2 cell lipids depends on cell differentiation

Previous studies from our laboratory have indicated that secondary hyperaldosteronism affects phospholipids of rat colonic enterocytes. To assess whether this represents a direct effect of mineralocorticoids on enterocytes, the role of aldosterone and dexamethasone in the regulation of lipid metabolism was examined in Caco-2 cells during development of their enterocyte phenotype. Differentiation of Caco-2 cells was associated with increased levels of triglycerides (TG) and cholesteryl esters (CE), a decreased content of cholesterol and phospholipids and changes in individual phospholipid classes. The phospholipids of differentiated cells had a higher content of n-6 polyunsaturated fatty acids (PUFA) and lower amounts of monounsaturated (MUFA) and saturated fatty acids than subconfluent undifferentiated cells. Differentiated cells exhibited a higher ability to incorporate [3H]arachidonic acid (AA) into cellular phospholipids and a lower ability for incorporation into TG and CE. Incubation of subconfluent undifferentiated cells with aldosterone or dexamethasone was without effect on the content of lipids, their fatty acids and [3H]AA incorporation. In contrast, aldosterone treatment of differentiated cells diminished the content of TG, increased the content of phospholipids and modulated their fatty acid composition. The percentage of n-6 and n-3 PUFA in phospholipids was increased and that of MUFA decreased, whereas no changes in TG were observed. The incorporation of [3H]AA into phospholipids was increased and into TG decreased and these changes were blocked by spironolactone. Treatment of differentiated cells with dexamethasone increased their CE content but no effect was identified upon other lipids, their fatty acid composition and on the incorporation of [3H]AA. As expected for the involvement of corticosteroid hormones the mineralocorticoid and glucocorticoid receptors were identified in Caco-2 cells by RT-PCR. The results suggest that aldosterone had a profound influence on lipid metabolism in enterocytes and that its effect depends on the stage of differentiation. The aldosterone-dependent changes occurring in phospholipids and their fatty acid composition may reflect a physiologically important phenomenon with long-term consequences for membrane structure and function.     

Phospholipid molecular species, beta-oxidation, desaturation and elongation of fatty acids in Atlantic salmon hepatocytes: effects of temperature and 3-thia fatty acids

We have investigated the effects of a 3-thia fatty acid (TTA) and of temperature on the fatty acid (FA) metabolism of Atlantic salmon (Salmo salar). One experiment investigated the activity of the peroxisomal beta-oxidation enzyme, acyl-CoA oxidase (ACO), and the incorporation of TTA into phospholipid (PL) molecular species. Salmon hepatocytes in culture were incubated either without TTA (control(spades)) or with 0.8 mM TTA (TTA(spades)) in a short term (48 h) temperature study at 5 degrees C and at 12 degrees C. TTA was incorporated into the four PL classes studied: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS). TTA was preferentially esterified with 18:1, 16:1, 20:4 and 22:6 in the PLs. Hepatocytes incubated with TTA had higher ACO activity at 5 degrees C than at 12 degrees C. In a second experiment salmon were fed a diet based on fish meal-fish oil without any TTA added (control) or a fish meal-fish oil diet supplemented with 0.6% TTA for 8 weeks at 12 degrees C and 20 weeks at 5 degrees C. At the end of the feeding trial, hepatocytes from fish acclimated to high or low temperatures were isolated from both dietary groups and incubated with either [1-(14)C]18:1 n-9 or [1-(14)C]20:4 n-3 at 5 degrees C or 12 degrees C. Radiolabelled 18:1 n-9 was mainly esterified into neutral lipids (NL), whereas [1-(14)C]20:4 n-3 was mainly esterified into PL at both temperatures. The rate of elongation of [1-(14)C]18:1 n-9 to 20:1 n-9 was twice as high in hepatocytes from fish fed the control diet than it was in hepatocytes from fish fed the TTA diet, at both temperatures. The amount of [1-(14)C]20:4 n-3 converted to 22:6 n-3 was approximately the same in hepatocytes from the two dietary groups, but there was a tendency to higher production of 22:6 n-3 at the lower temperature. Oxidation of [1-(14)C]18:1 n-9 to acid soluble products (ASP) and CO(2) was approximately 10-fold greater in hepatocytes kept at 5 degrees C than in those kept at 12 degrees C and the main oxidation products formed were acetate, oxaloacetate and malate.     

Incorporation of [3H]Arachidonic and [14C]Eicosapentaenoic Acids into Glycerophospholipids and Their Metabolism via Lipoxygenases in Isolated Brain Cells from Rainbow Trout Oncorhaynchus mykaiss

The incorporation of [3H]arachidonate [( 3H]AA) and [14C]eicosapentaenoate [( 14C]EPA) into glycerophospholipids was studied in isolated brain cells from rainbow trout, a teleost fish whose lipids are rich in (n-3) polyunsaturated fatty acids (PUFAs). EPA was incorporated into total lipid to a greater extent than AA, but the incorporation of both PUFAs into total glycerophospholipids was almost identical. The incorporation of both AA and EPA was greatest into phosphatidylethanolamine (PE). However, when expressed per milligram of individual phosphoglycerides, both AA and EPA were preferentially incorporated into phosphatidylinositol (PI), the preference being significantly greater with AA. On the same basis, significantly more EPA than AA was incorporated into phosphatidylcholine (PC). When double-labelled cells were challenged with calcium ionophore A23187, the 3H and 14C released from the cells closely paralleled each other, peaking at 10 min after addition of ionophore. The 12-monohydroxylated derivative was the pre-dominant lipoxygenase product from both AA and EPA with a rank order of 12-hydroxyeicosatetraenoic acid (12-HETE) greater than leukotriene B4 (LTB4) greater than 5-HETE greater than 15-HETE for the AA products and 12-hydroxyeicosapentaenoic acid (12-HEPE) greater than 5-HEPE greater than LTB5 greater than 15 HEPE for EPA products. The 3H/14C (dpm/dpm) ratios in the glycerophospholipids, total released radioactivity, and the lipoxygenase products suggested that PC rather than PI was the likely source of eicosanoid precursors in trout brain cells.     

12-Hydroxyeicosatetraenoic acid is metabolized by beta-oxidation in mouse peritoneal macrophages. Identification of products and proposed pathway

The products derived from the metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) by mouse peritoneal macrophages were characterized by high performance liquid chromatography (HPLC) and GC-mass spectrometry. HPLC analysis demonstrated two predominant polar products and several minor ones. The proportion and amounts of these products were dependent on the concentration of 12-HETE, the number of macrophages incubated with the monohydroxy fatty acid, and the time of incubation. The products identified by GC-mass spectrometry suggested that 12-HETE had undergone beta-oxidation. The intermediates identified were: 3,12-dihydroxy-5,8,10,14, 20:4; 10-hydroxy-3,6,8,12, 18:4; 3,10-dihydroxy-6,8,12, 18:3; 8-hydroxy-4,6,10, 16:3; 6-hydroxy-4,8, 14:2; and 4-hydroxy, 12:1. The major products, as identified by HPLC and GC-mass spectrometry, were 8-hydroxy-4,6,10, 16:3 and 4-hydroxy, 12:1. A minor product, 10-hydroxy-6,8,12, 18:3 was postulated to arise from either the isomerization and reduction of 10-hydroxy-3,6,8,12, 18:4 or from chain elongation of 8-hydroxy-4,6,10, 16:3. Inhibiting cyclooxygenase and lipoxygenase activities by ibuprofen and nordihydroguaiaretic acid, respectively, did not inhibit the formation of these products. 82% to 98% of 12-HETE was converted and released into the medium as products of beta-oxidation. The remainder was taken up into cellular lipids. beta-Oxidation of 12-HETE was decreased by only 12 and 21% after inhibiting mitochondrial fatty acid oxidation by 89 and 93% by 5 and 100 microM concentrations of the mitochondrial fatty acid oxidation inhibitor, methyl palmoxirate, respectively. It is thus postulated that the beta-oxidation of 12-HETE by mouse peritoneal macrophages occurs in peroxisomes.     

Effect of eicosapentaenoic acid on triacylglycerol transport in CaCo-2 cells

The human intestinal cell line, CaCo-2, was used to study the effect of the n-3 fatty acid, eicosapentaenoic acid, on triacylglycerol secretion. In cells incubated with 250 microM eicosapentaenoic acid, the incorporation of [3H]glycerol into triacylglycerols secreted into the medium was decreased by 58% compared to cells incubated with 250 microM oleic acid. The incorporation of [3H]glycerol into cellular triacylglycerols was decreased 32% in cells incubated with eicosapentaenoic acid. In cells preincubated with [3H]glycerol to label existing triacylglycerols, the rates of secretion of preformed triacylglycerols were similar in response to the addition of either fatty acid. Initial uptake rates of the n-3 fatty acid were higher than for oleic acid. Both eicosapentaenoic acid and oleic acid were minimally oxidized to CO2. Oleic acid was predominantly incorporated into cellular triacylglycerols (62% vs. 47%), whereas more eicosapentaenoic acid was incorporated into cellular phospholipids (46% vs. 30%). Phospholipids of microsomes prepared from cells incubated with eicosapentaenoic acid were enriched in this fatty acid. The rate of synthesis of triacylglycerol and diacylglycerol acyltransferase activities were significantly less in microsomes prepared from cells incubated with eicosapentaenoic acid. Triacylglycerol mass secreted by CaCo-2 cells incubated with either fatty acid was similar. In CaCo-2 cells, eicosapentaenoic acid decreases the synthesis and secretion of newly synthesized triacylglycerol without decreasing the secretion of triacylglycerol mass. Modification of microsomal membrane phospholipid fatty acid composition is associated with a decrease in microsomal triacylglycerol synthesis and diacylglycerol acyltransferase activities.     

Halothane metabolism. Impairment of hepatic omega-oxidation of leukotrienes in vivo and in vitro.

Omega-oxidation of leukotrienes is the initial step of hepatic degradation and thus inactivation of these proinflammatory mediators. Omega-oxidation is followed by beta-oxidation of leukotrienes from the omega-end. After exposure of rats to a single dose of the anesthetic agent halothane, a transient decrease in leukotriene omega-oxidation was induced both in vivo and in vitro. In untreated rats, 44.1 +/- 6.0% of N-[3H]acetylleukotriene E4 injected intravenously was recovered unchanged in bile collected for 60 min in vivo; 46.5 +/- 3.0% was recovered as omega-/beta-oxidation products, of which 24.7 +/- 4.5% were associated with beta-oxidation products only (mean +/- SEM; n = 5). In rats receiving a single dose of halothane 18 h before the experiment, recovery of unchanged N-[3H]acetylleukotriene E4 was significantly increased to 79.8 +/- 4.8%, while the fraction of omega-/beta-oxidation products decreased to 9.0 +/- 1.7% (n = 5); 90 h after exposure to halothane, N-[3H]acetylleukotriene E4 recovery decreased to 30.0 +/- 3.0% and omega-/beta-oxidation products amounted to 49.1 +/- 3.8%; the fraction of beta-oxidation products was significantly increased to 43.1 +/- 3.4% (n = 5). Ten days after exposure of rats to halothane, the recoveries of N-[3H]acetylleukotriene E4, of omega-/beta-oxidation products, and of beta-oxidation products alone, returned to almost normal values. Microsomal fractions obtained from rat hepatocytes catalyzed the NADPH- and O2-dependent leukotriene omega-oxidation in vitro. The formation of omega-hydroxy-metabolites of leukotriene B4, leukotriene E4, and N-acetylleukotriene E4 was decreased by 50% in microsomal fractions obtained from rats 18 h and 90 h after halothane treatment, and returned back to control levels in microsomal fractions obtained 10 days after halothane treatment. The Km value of leukotriene B4 omega-oxidation revealed no significant change in enzyme affinity towards leukotriene B4; in contrast, as reflected by the reduction of the Vmax value by 65%, a decrease in the amount of the active enzyme in microsomes obtained from rats 18 h after halothane treatment was observed. Halothane-metabolism-dependent trifluoroacetylation of hepatic proteins may mediate this process. Thus, the time course of the density on immunoblots of trifluoroacetylated protein adducts paralleled that of the transient decrease in leukotriene omega-oxidation. In contrast to its omega-oxidation, leukotriene B4 synthesis from 5-hydroperoxyeicosatetraenoate was not inhibited in hepatocyte homogenates obtained from rats pretreated with halothane. The data suggest that metabolism of halothane causes a transient derangement of hepatic leukotriene homeostasis in vivo.     

Blockade of Receptor-Mediated Cyclic GMP Formation by Hydroxyeicosatetraenoic Acid

Receptor-mediated cyclic GMP formation in N1E-115 murine neuroblastoma cells appears to involve oxidative metabolism of arachidonic acid. Evidence in support of this includes the blockade of this response by lipoxygenase inhibitors, e.g., eicosatetraynoic acid (ETYA) or other metabolic perturbants, e.g., methylene blue. It was recently discovered that the lipoxygenase products 15-hydroxyeicosatetraenoic (15-HETE) acid and 12-HETE, like ETYA, were inhibitors of M1 muscarinic receptor-mediated cyclic GMP formation. In the present report, the effects of monoHETEs are explored in more detail, particularly with regard to the function of the muscarinic receptor. Like 12-HETE and 15-HETE (IC50 = 13 and 11 microM, respectively), 5-HETE inhibited the cyclic GMP response to the muscarinic receptor (IC50 = 10 microM). All three of these monoHETEs were shown also to be inhibitors of the cyclic GMP responses to receptors stimulated by carbachol, histamine, thrombin, neurotensin, and bradykinin. 15-HETE was shown to inhibit the muscarinic receptor-mediated response in a complex manner (apparent noncompetitive and uncompetitive components; IC50 = 18 and 2 microM, respectively). 15-HETE did not inhibit either the M1 muscarinic receptor-stimulated release of [3H]inositol phosphates from cellular phospholipids or the M2 muscarinic receptor-mediated inhibition of hormone (prostaglandin E1)-induced AMP formation. It seemed possible that the monoHETEs could enter into biochemical pathways for arachidonate in N1E-115 cells. [3H]Arachidonate and the three [3H]-monoHETEs all rapidly labeled the membrane lipids of intact N1E-115 cells, with each [3H]eicosanoid producing a unique labeling profile. [3H]15-HETE labeling was noteworthy in that 85% of the label found in the phospholipids was in phosphatidylinositol (PI;t1/2 to steady state = 3 min). Exogenous 15-HETE inhibited the labeling of PI by [3H]arachidonate (IC50 = 28 microM) and elevated unesterified [3H]arachidonate levels. Thus, the mechanism of blockade of receptor-mediated cyclic GMP responses by monoHETEs is likely to be more complex than the simple inhibition of cytosolic mechanisms, e.g., generation of a putative second messenger by lipoxygenase, and may involve also alterations of membrane function accompanying the redistributions of esterified arachidonate.