Dihydroxyeicosatetraenoic Acids Research Articles

Enzymatic formation and regulatory function of lipoxins and leukotriene B4 in rat kidney mesangial cells.

Many metabolites of arachidonic acid (AA) have been demonstrated to act as second messengers for cellular functions and signal transduction. Following liberation from membrane phospholipids by the actions of phospholipases, arachidonic acid (AA) can be further metabolized by cyclooxygenase (CO), lipoxygenase (LO), and epoxygenase enzyme systems to generate oxygenated metabolites of AA that participate in the regulation of cell function. There are at least three lipoxygenases responsible for the generation of biologically active metabolites from AA. These are the 5-, 12-and 15-lipoxygenases, which give rise to the predominately pro-inflammatory leukotrienes (LTs) and the mono and di-hydroxy eicosatetraenoic acids (HETEs). Recently lipoxins, a class of structurally related trihydroxytetraenes derived from AA and eicosapentaenoic acid (EPA) have been described (1, 2). These compounds are formed by the sequential oxidation of these polyunsaturated fatty acids by 5-lipoxygenase and 15-Lipoxygenase or 12-Lipoxygenase (3,4). Lipoxins possess potent biological activities which are distinct from those of other lipoxygenase and cyclooxygenase products (5) and in many respects lipoxin have an apparent anti-inflammatory function. For example, prior exposure of neutrophils to LXA4, inhibits LTB4 induced phosphoinositol hydrolysis and impairs the chemotic response of polymorphonuclear cells to both LTB4 and formyl-methionyl-leucyl-phenylalanine (fMLP) (6).

Fatty acid composition and lipoxygenase metabolism in blood cells of the lesser spotted dogfish, Scyliorhinus canicula

1. 1. The fatty acid composition of erythrocytes and leucocytes of the elasmobranch, Scyliorhinus canicula, was determined so as to indicate substrate availability for eicosanoid formation. 2. 2. Leucocytes showed a greater degree of fatty acid unsaturation than the erythrocytes, with particularly high levels of docosahexaenoic acid (22:6,n−3). 3. 3. The major eicosanoid precursors, arachidonic acid (20:4,n−6) and eicosapentaenoic acid (20:5,n−3), represented 13.9% and 5.2% of the total fatty acid, respectively, in erythrocytes compared with 10.7% and 6% in leucocytes. 4. 4. Whole blood and isolated leucocytes were stimulated with calcium ionophore, A23187 and the resulting lipoxygenase products separated by reverse phase high performance liquid chromatography. 5. 5. The main lipoxygenase products formed were 6- trans-leukotriene B 4, 6- trans-12- epi-leukotriene B 4. 5(S),6(R) dihydroxyeicosatetraenoic acid and 5- and 15-hydroxyeicosatetraenoic acid. 6. 6. No leukotriene B 4, leukotriene B 5, or lipoxins were detected.

Formation of 8,15-dihydroxy eicosatetraenoic acidvia 15- and 12-lipoxygenases in fish gill.

Metabolites of arachidonic acid are important regulators of biological function in a variety of mammalian tissues. We have demonstrated similar lipoxygenase enzyme activities in fish gills and mammalian lungs suggesting that their metabolites may have matching functions. Fish gills were investigated for their ability to generate dioxygenated metabolites of polyunsaturated fatty acids. Fatty acids, including arachidonic acid, were incubated with crude tissue homogenates and polar metabolites were extracted, derivatized and analyzed by high performance liquid chromatography, gas chromatography and mass spectrometry. The major dihydroxy metabolite of arachidonic acid was characterized as 8(LR), 15(LS)-dihydroxyeicosatetraenoic acid. This product was formed by the sequential action of the 15- and 12-lipoxygenases in the tissue. The formation of the dihydroxyeicosatetraenoic acid by crude tissue homogenates was significantly enhanced by the addition of 1 mM reduced glutathione. The metabolism of other polyunsaturated fatty acids, including eicosapentaenoic acid and docosahexaenoic acid, to dihydroxy acids was consistent with their relative ability to serve as substrates for the initial 15-lipoxygenase reaction.

Conversion of dihomo-γ-linolenic acid to mono- and dihydroxy acids by potato lipoxygenase: Evidence for the formation of 8,9-leukotriene A 3

Evidence for the formation of a positional isomer of leukotriene (LT) C 3 (8,9-LTC 3) from dihomo-γ-linolenic acid has been published (Hammarström, S. J. Biol. Chem. 256, 7712–7714, 1981). This report describes the conversion of dihomo-γ-linolenic acid to a postulated intermediate in former reaction, 8,9-LTA 3, by purified lipoxygenase from potato tubers. 8( S)-Hydroperoxyeicosatrienoic acid (8( S)-HPETrE) was the most abundant dioxygenation product formed followed by 11-, 15-, and 12-HPETrEs (in decreasing order of abundance). In addition, 8( S),15( S)- plus 8( S), 15( R)-dihydroperoxyeicosatetraenoic acid (DiHPETrE) (EZE), and 8( S),15( S)- plus 8( S),15( R)-dihydroxy-eicosatetraenoic acid (DiHETrE) (EEE) were generated. Under anaerobic conditions only the latter two isomers of 8,15-DiHETrE (EEE) were obtained from 8-HPETrE. The results suggest that 8,9-LTA 3 is synthesized by the sequential action of 8- and 11-lipoxygenase activities associated with the potato enzyme.

Identification of a novel and highly potent eosinophil chemotactic lipid in human eosinophils treated with arachidonic acid.

Purified human eosinophils generate eosinophil chemotactic lipids (ECL), when incubated with arachidonic acid without any stimulus. Reversed phase HPLC of incubation supernatants revealed major lipid-like eosinophil chemotactic activity eluting in a peak containing 5(S), 15(S)dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid (5,15-DiHETE) as well as a 8,15-dihydroxyeicosatetraenoic acid. For further characterization of the ECL, some authentic dihydroxyeicosatetraenoic acids were tested for eosinophil chemotactic activity. Only 5,15-DiHETE as well as 8(S), 15(S)-dihydroxy-5,11-cis-9,13-trans-eicosatetraenoic acid were found to be chemotaxins for human eosinophils, however, with an ED50 near 0.3 microM and 1.5 microM, respectively. The presence of high titer eosinophil chemotactic activity in ECL preparations let us look for a contaminating ECL with higher specific activity. By using a different reversed phase HPLC-system 5,15-DiHETE as well as 8(R), 15(S)-dihydroxy-5,11-cis-9,13-trans-eicosatetraenoic acid could be separated from a highly potent ECL. Final purification of this ECL by the use of straight phase HPLC resulted in a single at 260 nm absorbing peak giving an UV spectrum different from that known for eosinophil chemotactic factors indicating a novel type of eosinophil chemotactic lipid. Eosinophil chemotactic activity of purified ECL has been found to be similar to that seen for platelet-activating factor, the most potent chemotaxin so far known, either in the number of migrating cells or the ED50. Cross-desensitization experiments with ECL, leukotriene B4, and platelet-activating factor revealed the existence of a separate ECL receptor on eosinophils. The production of potent ECL by the responder cells themselves supports the idea that there exists a self-sustaining mechanism of eosinophil accumulation.

Biochemistry of the lipoxygenase pathways in neutrophils

Three mammalian lipoxygenases have been reported to date. They catalyze the insertion of oxygen at positions 5, 12, and 15 of various 20-carbon polyunsaturated fatty acids. In the case of arachidonic acid, the immediate products are hydroperoxyeicosatetraenoic acids (HPETEs). HPETEs can undergo different transformations. One reaction is a reduction of the hydroperoxy group yielding the corresponding hydroxyeicosatetraenoic acids (HETEs). In the neutrophils, the major pathway of arachidonic acid metabolism is the 5-lipoxygenase. In these cells the 5-HPETE undergoes a cyclization reaction leading to a 5(6)-epoxy(oxido)eicosatetraenoic acid or leukotriene A4. The 5(6)-epoxy fatty acid can undergo three additional transformations: (a) a nonenzymatic hydrolysis to epimeric dihydroxyeicosatetraenoic acids (diHETEs); (b) stereospecific enzymatic hydrolysis to a specific diHETE, leukotriene B4; or (c) ring opening by reduced glutathione (GSH) to yield a peptidolipid, named leukotriene C4, in which GSH is attached via a sulfoether linkage. The leukotrienes constitute a group of biologically active substances probably involved in allergic and inflammatory reactions. The 5(6)-epoxy-eicosatetraenoic acid and the products derived from it contain a conjugated triene unit; the term leukotriene also denotes the cells (leukocytes) recognized to form these products, mainly the neutrophils, eosinophils, basophils, monocytes, mast cells, and macrophages. In the present article various aspects of the biochemistry of the lipoxygenase pathways of neutrophils are reviewed.

Enzymic Conversion of 11,12-Leukotriene A4 to 11,12-Dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic Acid: Purification of an epoxide hydrolase from the guinea pig liver cytosol

(11S,12S)-Epoxy-5,14-cis-7,9-trans-eicosatetraenoic acid (11,12-leukotriene A4) was nonenzymically converted to seven compounds: two diastereomers of (12S)-hydroxyeicosatetraeno-delta-lactones (major products), two diastereomers of (5,12S)-dihydroxyeicosatetraenoic acid and three stereoisomers of (11,12S)-dihydroxyeicosatetraenoic acid. Among these compounds, (11R,12S)-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid proved to be the only enzymic product. This hydrolysis activity was present in the cytosol fractions of various tissues of guinea pig such as liver, adrenal gland, small intestine, and brain. We purified the epoxide hydrolase to an apparent homogeneity from the guinea pig liver. The enzyme had a molecular weight of 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 7.3. The partial amino acid sequence was different from that of the microsomal enzyme. Km and Vmax values for 11,12-leukotriene A4 were 18 microM and 2.4 mumol/min/mg protein, respectively. These results indicate that 11,12-dihydroxyeicosatetraenoic acid is enzymically synthesized from 11,12-leukotriene A4 by the action of the cytosolic epoxide hydrolase in vitro.

Lipoxygenase activities of the epithelial cells of the human buccal cavity

The lipoxygenase activities of cultured human buccal epithelial cells and cells taken ex vivo from the human buccal cavity were compared. Lipoxygenation by cultured cells exhibited exclusively ω-6 positional specificity. A membrane-damaging event such as freezing was required for activation. In contrast, after simple addition of arachidonic acid, the ex vivo buccal cells produced predominantly 12-hydroxyeicosatetraenoic acid. Chromatography on chiral columns yielded products which had an (S) configuration at the site of oxygen insertion. 5-hydroxyeicosatetraenoic acid was transformed by ex vivo buccal cells to 5(S), 12(S)-dihydroxyeicosatetraenoic acid. These products could have a role in modulation of inflammatory states of the oral cavity.

The renal cytochrome P450 system generates novel arachidonic acid metabolites.

The generation of prostaglandins and other oxygenated metabolites of arachidonic acid (AA) is a complex process initiated by the release of ester-ified AA from cellular lipids. Once liberated from membrane lipids by diverse stimuli (peptide hormones, neurotransmitters and mechanical disruption), the free AA is rapidly metabolized. Metabolism of AA involves three pathways: (a) cyclooxygenase, leading to the formation of prostaglandins, thromboxane A2 (TxA2), and prostacyclin (PGI2); (b) lipoxygenases, leading to the formation of hydroxy- and dihydroxyeicosatetraenoic acids (HETEs and diHETEs) and leukotrienes; (c) cytochrome P450-dependent monoxy-genase system which metabolizes AA by an NADPH-dependent mechanism to a variety of oxygenated products such as HETEs, epoxyeicosatrienoic acids or epoxides (EETs) and their hydrolysis products, the dihydroxyeicosatrienoic acids or diols (DHTs) as well as ω and ω-1 hydroxylated acids (1–4). The pattern of AA metabolism in the kidney is distinct and AA metabolites participate in integrated renal function. Among the structures that metabolize AA via cyclooxygenase are collecting tubules (5), glomeruli (6), medullary interstitium (7) and blood vessels (8). Other structures such as the convoluted tubules have low or negligible cyclooxygenase activity (8). Lipoxygenase activity is mainly associated with leukocytes or platelets (9,10) and was reported to be present in isolated glomeruli (11).

Arachidonic acid 15-lipoxygenase and airway epithelium. Biologic effects and enzyme purification.

Pulmonary epithelial cells may be primarily responsible for initiating or regulating inflammatory responses in the airways, in part by releasing chemical mediators. Among the most potent mediators of inflammation are the lipoxygenase metabolites of arachidonic acid, including the leukotrienes and the other mono- and dihydroxyeicosatetraenoic acids (HETEs). The human airway epithelium contains significant 15-lipoxygenase activity. Although some biologic functions of 15-lipoxygenase metabolites are known, further understanding of the role of this enzyme in the airway requires localization in tissue and studies of expression, regulation, and biologic activity. Towards these aims, we purified and characterized 15-lipoxygenase from eosinophil-enriched leukocytes. First, we studied cofactors that may be involved in regulating enzymatic activity. We discovered that calcium and phosphatidylcholine both enhanced, but ATP inhibited, the 15-lipoxygenase activity of highly enriched enzyme. Second, we isolated to homeogeneity, for the first time, human 15-lipoxygenase. This led to the determination of the N-terminal amino acid sequence and the discovery of homology among various mammalian lipoxygenases. Further research using purified lipoxygenase is expected to increase our understanding of the biologic roles and biochemical features of 15-lipoxygenation of arachidonic acid.

Leukotriene A 4: metabolism in different rat tissues

The transformation of leukotriene A 4 into dihydroxyeicosatetraenoic acids and sulfidopeptide leukotrienes was determined in homogenates of rat tissues supplied with glutathione and albumin. The highest production of leukotriene B 4 was found in spleen, lung and small intestine, while leukotriene C 4 dominated in liver and lung. 5( S),6( R)-Dihydroxy-7,9- trans-11,14- cis-eicosatetraenoic acid (5,6-DHETE) was formed in all tissues, most prominently in kidney, heart and brain. We also found another isomer of 5,6-dihydroxyeicosatetraenoic acid produced in the kidney. This compound was derived from 5,6-DHETE by isomerization, probably of the 11-cis double bond to 11-trans, and the process appeared to be catalyzed by a membrane-bound factor.

Arachidonic acid metabolites from polymorphonuclear leukocytes of healthy donors, severely burned patients and children with cystic fibrosis—routine monitoring by high-performance liquid chromatography

A rapid and sensitive high-performance liquid Chromatographic method for the determination of arachidonic acid metabolites was developed. This method provides a clear and simple separation of the ω-oxidation products as well as of leukotriene B 4 and 5 S,12 S-dihydroxy-6- cis-8- trans-10- trans-14- cis-eicosatetraenoic acid. Furthermore, a solvent switch enables the detection of the monohydroxyeicosatetraenoic acids together with the cysteinyl-leukotrienes as well as leukotriene B 4 and the dihydroxyeicosatetraenoic acids within 70 min (including column equilibration for 10 min). The efficiency of the method for the routine monitoring of leukotrienes was performed with human polymorphonuclear leukocytes of healthy donors, severely burned patients and of children with cystic fibrosis. The advantages of this method include high sensitivity and easy handling, which are important for routine analyses of the arachidonic acid metabolites.

SK&F 86002: A structurally novel anti-inflammatory agent that inhibits lipoxygenase- and cyclooxygenase-mediated metabolism of arachidonic acid

The effects of SK&F 86002 [5-(4-pyridyl)-6 (4-fluorophenyl)-2,3-dihydroimidazo (2,1-b) thiazole] on the generation of eicosanoids in vitro and on inflammatory responses in vivo are described and compared to other non-steroidal anti-inflammatory drugs. SK&F 86002 inhibited prostaglandin H 2 (PGH 2) synthase activity ( ic 50 120 μM) as well as prostanoid production by rat basophilic leukemia (RBL-1) cells ( ic 50 70 μM) and its sonicate ( ic 50 100 μM) and human monocytes ( ic 50 1μM). In addition, SK&F 86002 inhibited the generation of dihydroxyeicosatetraenoic acid (diHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE) by a high speed supernatant fraction of RBL-1 cells ( ic 50 10 μM). Cellular production of 5-lipoxygenase products was inhibited by SK&F 86002 as measured by leukotriene B 4 (LTB 4) generation from human neutrophils ( ic 50 20 μM), leukotriene C 4 (LTC 4) generation by human monocytes ( ic 50 20 μM), and 5-HETE production by RBL-1 cells ( ic 50 40 μM). The in vivo profile of anti-inflammatory activity of SK&F 86002 supports the dual inhibition of arachidonate metabolism as indicated by its activity in inflammation models that are insensitive to selective cyclooxygenase inhibitors. The responses of arachidonic-acid-induced edema in the mouse ear and rat paw, as well as the cell infiltration induced by carrageenan in the mouse peritoneum and by arachidonic acid in the rat air pouch, were inhibited by SK&F 86002 and phenidone but not by the selective cyclooxygenase inhibitors naproxen and indomethacin.

Identification of blood leukotrienes in classical migraine.

SYNOPSISIn five patients affected by classical migraine a longitudinal study was performed, dealing with the in vitro parameters of platelet aggregation, during prodromal, attack, post‐attack and interval phases. These patterns were compared with the analysis of blood leukotrienes, carried out by use of analytical reversed phase HPLC. Presence of leukotriene C4 (LTC4) during prodromal and attack phases, corresponded to a transient inhibition of epinephrine‐induced aggregation. Recovery towards normal or increased values of platelet aggregation occurred during both post‐attack and interval phases, which were accompanied by the presence in blood of 5–12 di‐hydroxyeicosatetraenoic acid (5–12 DHETE) and/or leukotriene B4 (LTB4) respectively, and by their subsequent metabolic breakdown products. Hypotheses on the role of leukotrienes in the pathogenesis of migraine are discussed.

Differences in arachidonic acid release, metabolism and leukotriene B 4 synthesis in human polymorphonuclear leukocytes activated by different stimuli

Products of the 5-lipoxygenase pathway were analyzed after different stimuli in human polymorphonuclear leukocytes prelabeled with 3H-arachidonic acid. Upon stimulation with the Ca 2+ ionophore, A23187, polymorphonuclear leukocytes generate 118.2 ± 18 pg [ 3H]dihydroxyeicosatetraenoic acids (diHETEs, including 3H-leukotriene B 4 and its 6- trans-stereoisomeres), after exposure to serum coated zymosan (35.8 ± 9 pg) and N-fMet-Leu-Phe (39.5 ± 9 pg). Conversion of 3H-arachidonic acid paralleled its release after A23187 and fMet-Leu-Phe exposure leaving only 13.8 ± 7% and 13.6 ± 3% of the released 3H-arachidonic acid unmetabolized, respectively. In contrast, after stimulation with serum-coated zymosan only a small fraction of the released 3H-arachidonate was converted to 5-lipoxygenase products leaving 73.0 ± 5% of the released 3H-arachidonic acid unmetabolized. In parallel, leukotriene B 4 synthesis was studied in unlabeled polymorphonuclear leukocytes, resulting in 40 ± 15 ng upon A23187 stimulation, 4 ± 0.9 ng upon stimulation with fMet-Leu-Phe and 1.8 ± 0.9 ng after serum-coated zymosan, showing a different ratio of leukotriene B 4 to 3H-diHETE for A23187 in contrast to serum-coated zymosan and fMet-Leu-Phe. These results indicate that the coupling between the release of the precursor fatty acid and the metabolism via the 5-lipoxygenase pathway differs greatly between different stimuli.

Ethanol-enhanced transmembrane penetration of arachidonic acid and activation of the 5-lipoxygenase pathway in human leukocytes

Enhanced penetration by ethanol of exogenous arachidonic acid into human leukocyte preparations results in the production of large amounts of eicosanoids including 5-, 12- and 15-hydroxyeicosatetraenoic acids as well as the leukotrienes C 4 Δ 6- trans-leukotriene B 4, 12-epi-Δ 6- trans-leukotriene B 4, leukotriene B 4 and 5(S), 12(S)-dihydroxyeicosatetraenoic acid. The production of these compounds is affected by the concentrations of both ethanol and arachidonic acid independently in a complex manner with stimulation at lower concentrations and later relative inhibition. It was shown that the resulting leukotriene B 4 exhibited the same specific activity as exogenous arachidonic acid when labelled substrate was used.

Hyperalgesic properties of 15-lipoxygenase products of arachidonic acid.

Induction of hyperalgesia by leukotriene B4 (LTB4), a potent chemotactic factor for polymorphonuclear leukocytes (PMNLs), depends on the generation by cutaneous PMNLs of mediators that are probably derived from the 15-lipoxygenation of arachidonic acid. The capacity of dihydroxyeicosatetraenoic acid (diHETE) products of the 15-lipoxygenation of arachidonic acid in PMNL to elicit hyperalgesia was evaluated by assessing the effects of intradermal injection of synthetic diHETEs on the pressure nociceptive threshold in rats. (8R,15S)-Dihydroxyeicosa-(5E-9,11,13Z)-tetraenoic acid [(8R,15S)-diHETE] produced a dose-dependent hyperalgesia, as measured by decrease in threshold for paw withdrawal. The isomer (8S,15S)-diHETE antagonized in a dose-dependent manner this hyperalgesia due to (8R,15S)-diHETE but did not suppress prostaglandin E2-induced hyperalgesia. (8S,15S)-DiHETE produced a dose-dependent hypoalgesia, as reflected by an increase in nociceptive threshold, suggesting a contribution of endogenous (8R,15S)-diHETE to normal nociceptive threshold. The hypoalgesic effect of (8S,15S)-diHETE was blocked by corticosteroids but not by the cyclooxygenase inhibitor indomethacin. Neither (8R,15S)-dihydroxyeicosa-(5,15E-9,11Z)-tetraenoic acid nor (8R,15S)-dihydroxyeicosa-(5,11E-9,13Z)-tetraenoic acid exhibited any hyperalgesic or hypoalgesic activity. The stereospecificity of the effect of (8R,15S)-diHETE suggests that the induction of hyperalgesia is a receptor-dependent phenomenon and that (8S,15S)-diHETE may be an effective receptor-directed antagonist. The (8R,15S)-diHETE and (8S,15S)-diHETE from PMNL, keratinocytes, and other epithelial cells may modulate normal primary afferent function and contribute to inflammatory hyperalgesia.

Metabolism of [1- 14C] — arachidonic acid by cultured calf aortic endothelial cells: Evidence for the presence of a lipoxygenase pathway

Intact cultured calf aortic endothelial cells from a 10th – 14th subculture rapidly metabolize exogenous [1- 14C] - arachidonic acid by three different routes: i) incorporation into triglycerides and phosphilipids in a ratio of about 2:1, ii) formation of lipoxygenase metabolites (12-hydroxy-5,8,10,14-eicosatetraenoic acid) and iii) formation of cyclooxygenase metabolites (6-keto-PG F 1α and PG F 2α). From analyses by thinlayer chromatography and high-pressure liquid chromatography it was established that the main lipoxygenase metabolites in intact cells are 12-hydroxy-5,8,10,14-eicosatetraenoic acid and a compound proposed to be (a) dihydroxyeicosatetraenoic acid(s). In frozen and thawed cells the incorporation of arachidonic acid into cellular lipids is abolished, whereas the lipoxygenase pathway is strongly enhanced. Under these conditions the cells produce predominantly 15-hydroxy-5,8,11,13-eicosatetraenoic acid in addition to 12-hydroxy-5,8,10,14-eicosatetraenoic acid. The formation of lipoxygenase products was inhibited by heating the cells or by preincubation with nordihydroguaiaretic acid or BW 755°C, whereas indomethacin was without effect. The formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid by intact cells was inhibited by 5,8,11-eicosatriynoic acid, Indomethacin and acetylsalicylic acid inhibited the formation of cyclooxygenase metabolites. 15Ls-hydroxy-5,8,11,13-eicosatetraenoic acid was incorporated into cellular lipids, but not dihydroxyeicosatetraenoic acids. Exogenous [ 3H]-labelled prostacyclin and TxB 2 were not incorporated but were metabolized to less polar products.

Effect of K + depolarization on the synthesis of prostaglandins and hydroxyeicosatetra(5,8,11,14)enoic acids (HETE) in the rat retina : Evidence for esterification of 12-HETE in lipids

[ 14C]Arachidonic acid is metabolized to prostaglandins and hydroxyeicosatetraenoic acids in the rat retina. After intravitreal injection of [ 14C]arachidonic acid, 25% of the injected radiolabel was recovered in the retinal lipids. Phosphatidylcholine and phosphatidylinositol were most actively labeled; however, all glycerolipids incorporated arachidonic acid. The synthesis of prostaglandins E 2, F 2α, D 2, 6-keto-F 1α, thromboxane B 2 and hydroxyeicosatetraenoic acids was measured by high-performance liquid chromatography. The identity of 12-HETE was confirmed by gas chromatography-mass spectrometry. Incubation of prelabeled retinas in vitro promoted the release of [ 14C]arachidonic acid from glycerolipids. A 12-fold increase in the synthesis of hydroxyeicosatetraenoic acids occurred with no change in the synthesis of prostaglandins. Incubation in a depolarizing medium (45 mM K +) resulted in a selective increase in hydroxyeicosatetraenoic acids, an effect that was blocked by nordihydroguaiaretic acid (1 μM) and eicosatetraynoic acid (10 μM). 12-[ 3H 8]Hydroxyeicosatetraenoic acid, intravitreally injected, was incorporated into retinal lipids with a distribution similar to arachidonic acid. When retinas labeled with 12-[ 3H 8]hydroxyeicosatetraenoic acid were incubated, there was a large release of the incorporated radioactivity, and metabolism to other products with the Chromatographie properties of dihydroxyeicosatetraenoic acids. The release of 12-hydroxyeicosatetraenoic acid was not affected by depolarizing conditions (45 mM K +); however, the conversion of 12-hydroxyeicosatetraenoic acid to dihydroxy isomers was stimulated by K +. These experiments demonstrate active pathways for the generation of eicosanoids in the rat retina that are sensitive to membrane depolarization and lipoxygenase inhibitors.

Arachidonic acid metabolism by erythrocytes.

Rabbit, chicken, rat, and dog erythrocytes (10(9) cells/ml) synthesized immunologically active 12-hydroxyeicosatetraenoic acid (12-HETE) when stimulated by the Ca2+ ionophore, A-23187. The levels of immunologically active hydroxyeicosatetraenoic acid were independent of the number of white blood cells and platelets in the erythrocyte suspensions. Two products were resolved by high performance liquid chromatography; one product was identified as 12-HETE, while a second product appeared to be a dihydroxyeicosatetraenoic acid. Radiolabeled arachidonic acid was incorporated into phospholipids. Phosphatidylcholine and phosphatidylethanolamine were primary sources of the 12-HETE and dihydroxyeicosatetraenoic acid, all of which were released from the cells.