A novel eicosanoid is the major arachidonic acid metabolite of cultured human monocytes

Human peripheral blood monocyte-macrophages (M) generate a novel eicosanoid during in vitro culture. The metabolite is generated during incubation of the cells with ¹⁴C — arachidonic acid (AA). Lack of prior recognition of this metabolite probably results from the facts that: 1) on thin-layer chromatography (TLC) in two standard solvent systems, the novel metabolite co-chromatographed with either prostaglandin D₂ or thromboxane B₂, and 2) its generation, under the conditions studied, does not occur until between 90 and 180 minutes after culture initiaton which is a time period beyond that used for most leukocyte studies. The generation of the metabolite is inhibited by nordihydroguaiaretic acid (NDGA) but not by indomethacin. Base hydrolysis did not alter its migration on TLC. On both reversed phase and straight phase high pressure liquid chromatography (HPLC), the novel peak isolated by TLC elutes as a single major peak of radioactivity with a retention time different from the known leukotrienes, hydroxy acids, or their metabolites. Furthermore, the peak isolated on HPLC has a single ultraviolet absorption maximum at 270 nm. M cultured for 1 week prior to a 24 hour incubation with ¹⁴C-AA generated proportionally less of the novel eicosanoid (roughly 68% of total radiolabeled product) than did M cultured for 3 weeks prior to a similar incubation with ¹⁴C-AA (roughly 86% of total radiolabeled product). Under the conditions studied, the novel eicosanoid is the major AA metabolite generated from exogenous AA by cultured M and it appears to be generated in increasing quantity as the M differentiate.     

Synthesis of hydroxyeicosatetraenoic (HETEs) and epoxyeicosatrienoic acids (EETs) by cultured bovine coronary artery endothelial cells

Endothelial cells release several factors which influence vascular tone, leukocyte function and platelet aggregation. Some of these factors are metabolites of arachidonic acid, most notably prostacyclin. However, many of the endothelial metabolites of arachidonic acid have not been positively identified. The purpose of these studies is to identify the arachidonic acid metabolites synthesized by bovine coronary endothelial cells. Cultured bovine coronary artery endothelial cells were incubated with [ ¹⁴C]arachidonic acid. The incubation media was extracted and the radioactive metabolites resolved by a combination of reverse phase- and normal phase-high pressure liquid chromatography (HPLC). The cells synthesized 6-keto prostaglandin (PG)F_1α, PGE₂, 12-hydroxyheptadecatrienoic acid (HHT), 12-, 15-, and 11- hydroxyeicosatetraenoic acids (HETE), and 14,15-, 11,12-, 8,9-, and 5,6-epoxyeicosatrienoic acids (EET). Several of the HETEs were further analyzed by chiral-phase HPLC. The cells synthesized predominately 12(S)-, 15(S)-, and 11(R)-HETE. The synthesis of the S optical isomers of 12- and 15-HETE suggested that the 12- and 15-lipoxygenases were present in these cells. 11(R)-HETE is probably derived from cyclooxygenase. They also synthesized smaller amounts of 9-, 8- and 5-HETEs. The structures of the HETEs and EETs were confirmed by mass spectrometry. The release of 6-keto PGF_1α and 15-HETE was measured by specific radioimmunoassays. Melittin, thrombin, arachidonic acid and A23187 stimulated the release of both eicosanoids in a concentration-related matter. Under all conditions, the release of 6-keto PGF_1α exceed the release of 15-HETE. Therefore, cultured bovine coronary artery endothelial cells synthesize cyclooxygenase, lipoxygenase and cytochrome P-450 metabolites of arachidonic acid.     

Bio-synthesis of PGD2 and TXB2 by rabbit blood monocytes

A method for the preparation of a highly purified sample of rabbit blood monocytes is described. The metabolism of arachidonic acid (AA) in these cells was studied. Mononuclear cells were prepared by centrifugation on Ficoll-Paque gradients and the monocytes were obtained by further centrifugation and adherence onto plastic culture dishes. These procedures provided a preparation which contained 95% monocytes (non-specific esterase positive). Incubation of [1-¹⁴C]-AA with these cells produced four major metabolites which were separated by TLC; these corresponded to prostaglandin (PG) D₂, thromboxane (TX) B₂, 12-hydroxyheptadecatrienoic acid (HHT) and 12-/15- hydroxyeicosatetraenoic acid (HETE). A minor product which co-migrated with PGE₂ was also detected but neither 6-keto-PGF_1α nor PGF_2α were detected. Also, there was no evidence of the formation of 5-lipoxygenase products (5-HETE and LTB₄) by rabbit monocytes with or without calcium-ionophore A23187-stimulation. The production of PGD₂, TXB₂ and PGE₂ was further confirmed by analyzing [³H]-AA metabolites using high-performance liquid chromatography (HPLC) with tritiated standards as references. The biosynthesis of these compounds from endogenous substrate in A23187-stimulated monocytes was confirmed by specific radioimmunoassays with or without prior HPLC separation. The synthesis of immunoreactive LTB₄ and LTC₄ by A23187-stimulated cells was also monitored and found to be relatively low. The synthesis of PGD₂, TXB₂ and PGE₂ from both exogenous and endogenous substrate was suppressed by treatment of the monocytes with indomethacin (10⁻⁶ M).     

Ionophore-stimulated lipoxygenase activity and histamine release in a cloned murine mast cell, MC9

A cloned murine mast cell line designated MC9 expresses a 5-lipoxygenase activity when stimulated with the ionophore A23187. Upon addition of 0.5 uM ionophore, MC9 cells produce 270 ± 43 pmoles 5-HETE, 74 ± 40 pmoles 5,12 di HETEs and 65 ± 31 pmoles LTC₄/10⁶ cells from 37 uM exogenously added [1-¹⁴C]arachidonic acid in two minutes. 5-HETE and 5,12-di HETES, including LTB₄ were identified by GC/MS whereas LTC₄ was confirmed by HPLC mobility, bio-assay, RIA and enzymatic transformation. The principal cyclooxygenase products were PGD₂ and TxB₂ (8.5 ± 2.4 and 5.4 ± 1.2 pmoles/10⁶ cells respectively). Prostanoids were identified by comigration with authentic standards on two-dimensional thin layer chromatograms. Production of arachidonic acid lipoxygenase metabolites stimulated with ionophore proved relatively insensitive to removal of extracellular Ca⁺² and chelation by EGTA. In addition, MC9 5-lipoxygenase required only low micromolar amounts of exogenous arachidonic acid for maximal activity. Whereas production of arachidonic acid metabolites lasted only two to five minutes, histamine release stimulated with ionophore was not initiated until 5 minutes (12 ± 3% cellular histamine) and continued for 30 minutes (37 ± 7% cellular histamine). Although these cells metabolize arachidonic acid differently from the classic peritoneal-derived mast cell, they resemble subpopulations found in certain tissues (such as mucosa) and should be useful in understanding the biochemistry of mast cell mediator release.     

Metabolism of exogenous arachidonic acid by murine macrophage-like tumor cell lines

Murine macrophage-like cell lines, J774.2, P388D1, RAW264.7 and PU-5-1R, were incubated with exogenous arachidonic acid (AA). The major metabolites were identified by comigration with known standards in TLC and HPLC and by characteristic behavior following reduction. During a 30 min incubation J774.2 cells metabolized exogenous ¹⁴C-AA (10 μM) to PGE₂ (14.8%), 12-hydroxy-5,8,10-heptadecatrienoic acid (HTT)_ (13.0%), thromboxane B₂ (TXB₂) (7.4%), PGD₂ (4.4%) and PGF_2α (3.0%). The remainder was incorporated into phospholipids (39.0%), triglycerides (6.1%), and as yet unidentified metabolites (8.2%). No PGF_1α was found. Metabolism of exogenous AA was rapid, being >90% completed at 3.5 min. Metabolism of exogenous AA is not increased by the simultaneous addition of macrophage stimuli including the cation ionophore A-23187, particulate phagocytic stimuli and endotoxin. The synthesis of cyclooxygenase products was inhibited by low doses of indomethacin (ID₅₀=0.6 μM) while the synthesis of TXB₂ and HHT was selectively inhibited by benzylimidazole (ID₅₀=9.5 μM). Identification of a probable lipoxygenase product is being pursued. The synthesis of this product is not inhibited by indomethacin and migrates with an R_f value close to 5,12-diHETE in TLC. P388D1 and RAW264.7 cells metabolize exogenous AA to the same products as J774.2, in different proportions, while PU-5-1R does not produce cylooxygenase metabolites to any appreciable extent.     

Early alterations induced by carbon tetrachloride in the lipids of the membranes of the endoplasmic reticulum of the liver cell II. Distribution of the alterations in the various lipid fractions

The distribution of carbon tetrachloride-induced alterations of membrane lipids in various fractions of liver microsomal lipids was studied. The chromatographic spot (referred to as the “D” spot in the previous paper [1]) which has been shown to contain the compounds responsible for the diene conjugation absorption [1], was found in the fatty acid methyl esters prepared from the fraction containing phosphatidylethanolamine (PE) and also in those obtained from the fraction containing phosphatidylserine (PS) and phosphatidylinositol (PI). The absorption of conjugated dienes was very marked in PE and less intense in PS and PI. The fatty acid methyl esters prepared from the fraction containing phosphatidylcholine (PC) showed no presence of the “D” spot and minimal absorption of conjugated dienes.A decrease in arachidonic acid content was found in the fraction containing PE, while no change in content of this fatty acid was found in the fraction containing PC. Results similar to those observed for PC were also found for neutral lipids (NL).Analysis of the fatty acid methyl esters of the various lipid fractions by gas-liquid chromatography (GLC) with an electron capture detector (ECD) gave a qualitative index of the free radical attack by CCl₄ metabolites. Quantitative estimation was attained by study of the irreversible binding of ¹⁴C from ¹⁴CCl₄ to the various lipid fractions. It was found that the fraction containing PS had the highest specific activity, while the fraction containing PC had the lowest specific activity of all the phospholipids. Thin layer chromatography (TLC) of the fraction containing PS revealed that only 11% of the radioactivity was associated with the pure PS moiety, while the remainder was associated with uncharacterized lipids (probably oxidation products).The possible relevance of the alterations induced by carbon tetrachloride in the various phospholipid fractions of liver microsomes to functional changes is discussed.     

An imbalance of arachidonic acid metabolism in asthma

Metabolism of arachidonic acid via the cyclooxygenase and lipoxygenase pathways was studied in washed platelets from normal and asthmatic subjects. The platelets were incubated with [1-¹⁴C] arachidonic acid and the metabolites formed were separated by high pressure liquid chromatography (HPLC). The platelets from asthmatic patients had a 40% decrease in cyclooxygenase-derived metabolites and a 70% increase in lipoxygenase-derived product when compared with metabolites generated by platelets from normal subjects. The ratio of cyclooxygenase to lipoxygenase products was 3.24 ± 0.26 for platelets from normal subjects, and 1.14 ± 0.15 with platelets from the asthmatic patients. These results indicate an imbalance of arachidonic acid metabolism in platelets from asthmatic patients.     

The identification of hydroxy fatty acids in psoriatic skin

Psoriasis is a common chronic inflammatory and proliferative skin disease characterised by epidermal neutrophil infiltration which may be induced by chemotactic substances in the involved epidermis. Superficial psoriatic scale was shown to contain biologically active amounts of leukotriene B₄ and monohydroxy-eicosatetraenoic acid (HETE)- like material as determined by assay for chemokinetic activity in high performance liquid chromatography (HPLC) fractions of scale extracts. Extracts of scale and chamber fluid from abraded lesional and uninvolved psoriatic skin were purified by HPLC and appropriate fractions were analysed by gas chromatography - mass spectrometry (GC-MS). The following monohydroxy metabolites of arachidonic, linoleic and 11,14-eicosadienoic acids were identified : 15-HETE, 12-HETE, 11-HETE, 9-HETE, 8-HETE, 5-HETE, 13-hydroxy-octadecadienoic acid (13-HODD), 9-HODD and 15-hydroxy-eicosadienoic acid (15-HEDE). The results suggested that 12-HETE, 13-HODD and 9-HODD are the most abundant monohydroxy fatty acids in the psoriatic skin extracts described above. Assays of 13-HODD, 9-HODD and 15-HEDE for chemokinetic activity were negative with concentrations up to 10^?4M. The biological significance of these three compounds in not known, but some of the hydroxylated metabolites of arachidonic acid may, by virtue of their chemotactic properties, be relevant to the pathogenesis of the psoriatic neutrophil infiltrate.     

Metabolism of Arachidonic Acid to Epoxyeicosatrienoic Acids. Hydroxyeicosatetraenoic Acids, and Prostaglandins in Cultured Rat Hippocampal Astrocytes

Abstract: We have recently shown that brain slices are capable of metabolizing arachidonic acid by the epoxy-genase pathway. The purpose of this study was to begin to determine the ability of individual brain cell types to form epoxygenase metabolites. We have examined the astrocyte epoxygenase pathway and have also confirmed metabolism by the cyclooxygenase and lipoxygenase enzyme systems. Cultured rat hippocampal astrocyte homogenate, when incubated with radiolabeled [³H]-arachidonic acid, formed products that eluted in four major groups designated as R_17–30, R_42–50, R_51–82, and R_83–90 based on their retention times in reverse-phase HPLC. These fractions were further segregated into as many as 13 peaks by normal-phase HPLC and a second reverse-phase HPLC system. The principal components in each peak were structurally characterized by gas chromatography/electron impact-mass spectrometry. Based on HPLC retention times and gas chromatography/electron impactmass spectrometry analysis, the more polar fractions (R_17–30) contained prostaglandin D₂ as the major cyclooxygenase product. Minor products included 6-keto prostaglandin F_1α, prostaglandin E₂, prostaglandin F_2α, and thromboxane B₂. Fractions R_42–50, R_51–82. and R_83–90 contained epoxygenase and lipoxygenase-like products. The major metabolite in fractions R_83–90 was 5, 6-epoxyeicosatrienoic acid (EET). Fractions R_51–82 contained 14, 15-and 8, 9-EETs, 12-and 5-hydroxyeicosatetraenoic acids, and 8, 9-and 5, 6-dihydroxyeicosatrienoic acids (DHETs). In fractions R_42–50, 14, 15-DHET was the major product. When radiolabeled [³H]14, 15-EET was incubated with astrocyte homogenate, it was rapidly metabolized to [³H]14, 15-DHET. The metabolism was inhibited by submicromolar concentration of 4-phenylchalcone oxide, a potent inhibitor of epoxide hydrolase activity. Formation of other polar metabolites such as triols or epoxyalcohols from 14, 15-DHET was not observed. In conclusion, astro-cytes readily metabolize arachidonic acid to 14, 15-EET, 5, 6-EET, and their vicinal-diols. Previous studies suggest these products may affect neuronal function and cerebral blood flow.     

Formation of leukotrienes and hydroxy acids by human neutrophils and platelets exposed to monosodium urate

Monosodium urate (MSU) crystals stimulate the production of arachidonic acid metabolites by human neutrophils and platelets. Neutrophils exposed to MSU generated leukotriene B₄(LTB₄). 6- -LTB₄, 12- -6- -LTB₄, and 5S, 12S DHETE from endogenous sources of arachidonate. In addition to these metabolites both monohyroxyeicosatetraenoic acids (i.e., 5-HETE) and w-oxidation products (i.e., 20-COOH LTB₄) were formed by neutrophils exposed to MSU. Addition of exogenous arachidonic acid led to increased formation of each of these metabolites. When neutrophils were treated with colchicine (10 uM), LTB₄ but 5-HETE formation was impaired. (1-¹⁴C) Arachidonate-labeled platelets exposed to MSU released (1-¹⁴C)-arachidonate. (¹⁴C)-12 HETE, (¹⁴C)-HHT and (¹⁴C)-thromboxane B₂. Results indicate that MSU stimulates arachidonic acid metabolism in both human neutrophils and platelets. Moreover, they suggest not only that metabolites of arachidonate may be considered as possible candidates for mediators of inflammation in crystal-associated diseases, but that colchicine blocks the formation of LTB₄.     

Hydroxylation of prostaglandin E1 by kidney cortex microsomal monooxygenase in the guinea pig

The incubation of [5,6-³H]prostaglandin E₁ ([³H]PGE₁) with guinea pig kidney cortex microsomes in the presence of NADPH in an atmosphere of air, resulted in chromatographically polar metabolites. The incubation products were treated with base which converted PGE₁ derivatives into PGB₁ derivatives, with a λ_max = 278 nm and the products were analyzed by TLC and high pressure-liquid chromatography (HPLC). Based on UV absorption, mobility on TLC and retention time in HPLC, as compared with authentic compounds, it was concluded that the two polar UV-absorbing peaks in HPLC represented 19-hydroxy-PGB₁ (19-OH-PGB₁) and 20-hydroxy-PGB₁ (20-OH-PGB₁). Further identification of the metabolites was obtained by derivatizing the incubation products as methyl esters and t-butyldimethylsilyl ethers, followed by co-injection with similarly derivatized authentic compounds in HPLC and gas chromatography. Finally, the derivatized metabolites were identified by comparing their mass fragmentation with that of similarly derivatized authentic compounds. There was an absolute requirement for NADPH, and NADH did not significantly support the hydroxylation of PGE₁. Inhibitors of microsomal monooxygenase (SKF 525A, metyrapone, and cytochrome c) inhibited the hydroxylation of PGE₁ by kidney cortex microsomes. By contrast, carbon monoxide at a CO:O₂ ratio of 5:1 did not inhibit the hydroxylation of PGE₁, pointing to a low or lack of CO sensitivity of the hydroxylation of PGE₁. The addition of PGE₁ or laurate to guinea pig kidney cortex microsomes elicited Type I spectral changes. The spectral dissociation constant (K_s) for PGE₁ was 2.4 × 10^?4m. The kinetic constants for 19- and 20-hydroxylations of PGE₁ were determined. The K_M values for the 19- and 20-hydroxylation pathways were found to be identical, being 3.3 × 10^?4m, suggesting that the same enzyme is involved in both hydroxylations; however, the V_max values for 19-hydroxylation and 20-hydroxylation of PGE₁ were 50 nmol/hr and 20.8 nmol/hr respectively. These results demonstrate that PGE₁ is a substrate for the kidney cortex microsomal monooxygenase. The similarities and differences of the kidney monooxygenase in the guinea pig with that in the rat are discussed.     

The use of high pressure liquid chromatography (HPLC) for the separation of radiolabeled arachidonic acid and its metabolites produced by thrombin-treated human platelets: II. Establishment of optimal assay conditions

We have utilized HPLC to develop optimal conditions for assaying the transformation of arachidonic acid in thrombin-treated human platelets. In the presence of increasing amounts of albumin, the total amount of radioactivity released from thrombin-treated platelets pre-labeled with ³H-arachidonic acid is first enhanced and then inhibited. Maximal release, reflecting primarily enhanced amounts of free labeled arachidonic acid, occurs at a final albumin concentration of 0.5 mg/ml. Calcium promoted the release of all radiolabeled metabolites, but it specifically enhanced HETE formation and release. Magnesium was without effect. Cyclo-oxygenase derived products constituted the bulk of released label at short time intervals, but after ten minutes exposure to thrombin in the presence of albumin (0.5 mg/ml) and 3 mM calcium, radioactivity in the released products was equally distributed among cyclo-oxygenase derived products (TXB₂ + PGD₂ + HHT), HETE and free arachidonic acid.     

The use of high pressure liquid chromatography (HPLC) for the separation of radiolabeled arachidonic acid and its metabolities produced by thrombin-treated human platelets: I. The validation of the technique

We have developed a technique for the rapid separation and quantitative collection of thromboxane B₂ (TXB₂), PGE₂, PGD₂, PGF_2α, 12-hydroxy-5,8,10 heptadecatrienoic acid (HHT), 12-L-hydroxy-5,8,10,14 eicosatetraenoic acid (HETE), and arachidonic acid released from thrombin treated human platelets. Platelets were pre-labeled with ³H-arachidonic acid and then isolated by gel filtration. They were then exposed to thrombin for various intervals and separated by centrifugation. Aliquots of the cell-free medium were applied directly to a high pressure liquid chromatograph containing a fatty acid column as the stationary phase. A quarternary solvent system containing tetrahydrofuran (THF), acetonitrile (CH₃CN), water and acetic acid (HOAC) resolved and eluted the arachidonic acid metabolites within 30 minutes. Since no sample preparation is required and since the solvent system does not quench the counting efficiency of a standard liquid scintillation fluor the technique permits rapid separation and quantitation of radiolabeled arachidonic acid and its metabolites.     

Prostaglandin-mediated inhibition of noradrenaline release: III. Separation of prostaglandins released from stimulated hearts and analysis of their neurosecretion inhibitory capacity

Isolated rabbit hearts were infused with ¹⁴C-arachidonic acid and subjected to sympathetic nerve stimulation. Prostaglandins in the cardiac effluent were extracted and separated using thin layer chromatography. Other hearts were infused with un-labelled arachidonic acid and the effluent was assayed for neurosecretion inhibitory capacity on the field-stimulated guinea pig vas deferens, and for anti-aggregatory activity on ADP-induced platelet aggregation. PGs in the effluent from hearts infused with un-labelled arachidonic acid were extracted and separated on TLC, and the different fractions were assayed for neurosecretion inhibitory activity.Sympathetic nerve stimulation after preincubation with ¹⁴C-AA elicited outflow of four different peaks of ¹⁴C-labelled PGs: one chromatographing close to PGF_2α (probably mainly 6-keto-PGF_1α), and three peaks corresponding to PGA₂/PGB₂, PGD₂, and PGE₂ respectively. The cardiac interstitial effluent contained anti-aggregatory material which was inactivated by heat treatment, and thus probably identical to PGI₂. The cardiac effluent also contained material with neuro-secretion inhibitory activity, which was resistant to heat treatment. Fractional assay of the TLC separated cardiac effluent demonstrated that the neurosecretion inhibitory activity chromatographed with PGE₂ only.It has earlier been observed that endogenous PGs inhibit trans-mitter release in sympathetically stimulated organs. On the basis of the current data we suggest that PGE₂ is the only physiological inhibitor of sympathetic transmitter release.     

Single step thin-layer chromatographic method for quantitation of enzymatic formation of fatty acid anilides

The activity of the enzyme involved in catalyzing the formation of fatty acid anilides can be measured by quantitating the fatty acid anilides formed. We have shown earlier that oleic acid is the most preferred substrate among other fatty acids studied for the conjugation with aniline. The reaction product (oleyl anilide) could be separated by thin-layer chromatography (TLC) and then quantified by reversed-phase high-performance liquid chromatography (HPLC). Using [1-¹⁴C]oleic acid as substrate, the fatty acid anilide forming activity can be determined in a single step by TLC analysis. The conventional TLC methods used for the separation of the fatty acid esters, however, could not resolve oleyl anilide from the residual [1-¹⁴C]oleic acid. Therefore, a simple and reliable TLC method was developed for the separation of oleyl anilide from oleic acid using a freshly prepared solvent consisting of petroleum ether–ethyl acetate–ammonium hydroxide (80:20:1, v/v). Using this solvent system the relative flow (R_f) values were found to be 0.54 for oleyl anilide and 0.34 for aniline, whereas oleic acid remained at the origin. The TLC procedure developed in the present study could be used to determine the fatty acid anilide forming activity using [1-¹⁴C]oleic or other fatty acids as substrate and was also found suitable for the analysis of fatty acid anilides from the biological samples.     

Transformation of arachidonic acid in the rat anterior pituitary

Rat anterior pituitaries were incubated with [1-¹⁴C]-arachidonic acid. The metabolites were purified by reversed-phase high pressure liquid chromatography. Conclusive identification of the compounds was performed by gas chromatography-mass spectrometry. The major metabolite of arachidonic acid was the 12-hydroxy-5,8,10,14-icosatetraenoic acid (0.1% of added radioactivity). Smaller amounts of 12-hydroxy-5,8,10-heptadecatrienoic acid and of 15-hydroxy-5,8,11,13-icosatetraenoic acid (0.01% of added radio-activity) were also isolated. Trace amounts of prostaglandins E₂, D₂ and F₂α were detected.     

Involvement of phospholipase A2 and arachidonic acid in the depolarization-evoked accumulation of Ca2+ in hippocampal mossy fiber nerve endings

Depolarization-evoked increases in intraterminal free Ca²⁺ are required for the induction of neurotransmitter release from nerve terminals. Although the mechanisms that regulate the voltage-induced accumulation of presynaptic Ca²⁺ remain obscure, there is evidence that the phospholipase-dependent accumulation of arachidonic acid, or its metabolites, may be involved. Therefore, fura-2 loaded hippocampal mossy fiber nerve endings were used to investigate the relationships between membrane depolarization, lipid metabolism and presynaptic Ca²⁺ availability. It was observed that depolarization of the nerve terminals with KCl induced an increase in intraterminal free calcium that was inhibited more than 90% by a combination of voltage-sensitive Ca²⁺ channel blockers. In addition, the K⁺-dependent effects on Ca²⁺ concentrations were attenuated in the presence of phospholipase A₂ inhibitors, but were mimicked by the phospholipase A₂ activator melittin and exogenous arachidonic acid. Both the melittin- and arachidonic acid-induced increases in presynaptic Ca²⁺ were reduced by voltage-sensitive Ca²⁺ channel blockers. The stimulatory effects of arachidonic acid appeared to be independent of its further metabolism to prostaglandins. In fact, inhibition of either cyclooxygenase or lipoxygenase pathways resulted in a potentiation of the depolarization-evoked increase in intraterminal free Ca²⁺. From these results, we propose that some portion of the depolarization-evoked increase in intraterminal free calcium depends on the activation of phospholipase A₂ and the subsequent accumulation of unesterified arachidonic acid.     

Biochemical interaction of arachidonic acid and vitamin e in human platelets

The effect of α-tocopherol on the oxidative transformation of arachidonic acid was investigated in human platelets. The major products of lipoxygenase and cyclo-oxygenase pathways were separated by high performance liquid chromatography (HPLC) and thin layer chromatoggaphy (TLC) evaluated by scanning the radiochromatograms. This study differs from others in the vitamin E field in important aspects of its experimental design: the prelabeling of platelets with non-aggregating concentrations of ¹⁴C-arachidonic acid, and the addition of α-tocopherol as a colloidal suspension rather than as an ethanolic solution. A moderately potent but consistent reduction of apparent cyclo-oxygenase activity by α-tocopherol could be demonstrated by TLC and HPLC. This effect was best shown by the change of the HETE/HHT ratio which increased significantly in vitamin E-treated platelets. It was found to be dosedependent up to 1 MM a-tocopherol, the maximal concentration tested in this study. Alpha-tocopherol quinone was equally effective in this action.     

Metabolic products of arachidonic acid in rats

The pattern of eicosanoid metabolites appearing in urine and feces following oral administration of radioactive arachidonic acid was investigated using rats deficient in essential fatty acids. About 70–80% of the radioactivity in the urine during the first day after feeding was adsorbed to XAD-2 resin and he represented eicosanoid metabolites, whereas the rest of the radioactivity was mainly ³H₂O. The eicosanoid metabolites were fractioned into different polarity classes using reverse phase Sep-Pak C₁₈ cartridges. Gas chromatographic analysis of the urinary metabolites following their derivatization into methyl ester-methoxime- -butyl-dimethylsilyl ethers revealed that nearly one-half of the metabolites had ECL values less than 22 and represented metabolites more oxidized than commonly described. Only 30% of the metabolites had ECL values between 26 to 32, corresponding to the values for the metabolites that originate from exogenously infused prostaglandins. A large portion of the eicosanoid metabolites was also excreted with the feces. The isotropic patterns from the reverse phase chromatography indicated that many of the fecal metabolites may be similar to those in urine although some metabolites in feces were not present in urine. Based on the specific radioactivity of the administered arachidonic acid, it appeared that at least 6 to 8 mg of eicosanoid metabolites were excreted through urine and feces within 24 hrs following an oral bolus of 60 mg arachidonic acid. The rapid increase and subsequent decrease in eicosanoid metabolite excretion after oral administration of arachidonate indicates that the dietary intake of polyunsaturated fatty acids may have a more rapid effect upon the endogenous production of eicosanoids than is generally recognized.     

Metabolism of arachidonic acid by isolated rat hepatocytes,renal cells and by some rabbit tissues: Detection of vicinal diols by mass fragmentography

Purified cytochromes P-450 (LM₂ and PB-B₂) in a reconstituted system and epoxide hydrolase were recently found to metabolize arachidonic (eicosatetraenoic) acid to four vicinal dihydroxyeicosatrienoic acids. These metabolites were chemically synthetized from octadeuterated arachidonic acid and employed as internal standards for mass fragmentography. Isolated rat hepatocytes and renal cells were incubated with arachidonic acid (0.1 mM; 37°C, 15 min) and, following extractive isolation and reversed-phase HPLC, formation of 11,12-dihydroxy-5,8,14-eicosatrienoic acid and 14,15-dihydroxy-5,8,11-eicosatrienoic acid was demonstrated by mass fragmentography using a capillary GC column. Furthermore, these diols were also detected in rabbit liver and renal cortex and they therefore appear to be formed endogenously. Formation of vicinal diols was also studied in cell free systems. Rabbit liver and renal cortical microsomes were incubated with NADPH (1 mM) and arachidonic acid (0.15 mM) for 15 min at 37°C and, besides 11,12-dihydroxy- and 14,15-dihydroxyeicosatrienoic acid, small amounts of 8,9-dihydroxy- and 5,6-dihydroxyeicosatrienoic acid could be detected by mass fragmentography. Renal as well as hepatic monooxygenases can thus epoxidize each of the four double bonds of arachidonic acid. In contrast, rabbit lung microsomes and NADPH metabolize arachidonic acid mainly to prostaglandins and 19-hydroxy- and 20-hydroxyarachidonic acid, while only small amounts of 11,12-dihydroxyeicosatrienoic acid could be found. Monooxygenase metabolism of arachidonic acid by epoxidation might therefore be a significant pathway for the metabolism of this essential fatty acid in isolated rat renal cells and hepatocytes but presumably not in the lung.