Cytochalasins inhibit arachidonic acid metabolism in thrombin-stimulated platelets.

Low concentrations (0.5-1 microM) of cytochalasins inhibit the thrombin-stimulated polymerization of monomeric actin to filamentous actin in platelets. Similar concentrations of cytochalasin B inhibit the formation and metabolism of arachidonic acid in horse platelets stimulated by low concentrations of thrombin (0.1-0.5 unit/ml). However, the release of serotonin is not inhibited by cytochalasin B. Cytochalasins B and D (0.5-1 microM) markedly reduce, in thrombin-stimulated human or horse platelets, the metabolism of the liberated arachidonic acid by cyclooxygenase activity to thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid and the conversion of arachidonic acid by lipoxygenase activity to 12-hydroxy-5,8,10,14-icosatetraenoic acid. The generation of arachidonic acid from platelet phospholipids and the formation of phosphatidic acid are much less affected by cytochalasin B or D. Cytochalasins do not directly inhibit platelet cyclooxygenase, lipoxygenase, phospholipase A2, or phosphatidyl-inositol-specific phospholipase C. In addition, the metabolism of exogenously added arachidonic acid by intact platelets is not inhibited by cytochalasins B and D. The results indicate that polymerization of actin in platelets stimulated by thrombin may be required for the effective metabolism of arachidonic acid released from platelet phospholipids.     

Arachidonic acid metabolism in polymorphonuclear leukocytes: Effects of ionophore A23187*

Addition of arachidonic acid and the divalent cation ionophore A23187 to a suspension of human peripheral blood polymorphonuclear leukocytes led to the formation of (5S)-hydroxy-6,8,11,14-icosatetraenoic acid, (15S)-hydroxy-5,8,11,13-icosatetraenoic acid, and (5S,12R)-dihydroxy-6,8,10,14-icosatetraenoic acid. A method based on high-pressure liquid chromatography has been developed for assay of these metabolites. The addition of arachidonic acid to human polymorphonuclear leukocytes always resulted in formation of the isomeric monohydroxy acids. However, cells prepared from blood of different subjects were found to vary with respect to formation of the 5,12-dihydroxy acid. Addition of the ionophore alone strongly stimulated the formation of the 5-monohydroxy acid and more specifically the 5,12-dihydroxy acid from endogenous arachidonic acid. In all experiments performed the formation of the 5-hydroxy acid and the 5,12-dihydroxy acid was maximally stimulated when both arachidonic acid and the ionophore were added to the incubation mixture. Under these conditions, stimulation of 40-fold or more of the formation of both compounds was observed. The data demonstrate that, in addition to causing release of endogenous substrate, the ionophore also activated the enzymatic system involved in the further transformations of arachidonic acid. This finding raises the possibility that this pathway of arachidonic acid metabolism is involved in the biological response (e.g., release of lysosomal enzymes, the slow reacting substance of anaphylaxis, and chemotactic factors) of leukocytes to A23187 and other stimuli.     

A second pathway of leukotriene biosynthesis in porcine leukocytes.

Incubation of suspensions containing polymorphonuclear and eosinophilic leukocytes with arachidonic acid led to the formation of two pairs of diastereomeric 8,(15S)-dihydroxy-5,9,11,13-icosatetraenoic acids and two erythro-14,15-dihydroxy-5,8,10,12-icosatetraenoic acids. The structures were elucidated by ultraviolet spectroscopy and gas chromatography--mass spectrometric analysis of several derivatives of each compound, catalytic hydrogenation, oxidative ozonolysis with steric analysis of alcohols, and comparison to reference compounds prepared by chemical synthesis. Experiments with 18O2 and H218O indicated that in all six compounds the hydroxyl group at C-15 was derived from molecular oxygen. Two of the diastereomeric 8,15-dihydroxy acids incorporated H218O at C-8, while the other two 8,15-dihydroxy products (also diastereomers) and the 14,15-dihydroxy compounds (geometric isomers) incorporated 18O2 at C-8 and C-14, respectively, in addition to C-15. Two of the 8,15-dihydroxy acids are formed by reaction of water with an unstable allylic epoxide intermediate, (14S,15S)-oxido-5,8,10,12-icosatetraenoic acid; the two 14,15-dihydroxy acids are proposed to be formed by reaction of activated molecular oxygen with the same epoxide, which in turn originates via 15S oxygenation of arachidonic acid.     

Metabolism of arachidonic acid by rat adrenal glomerulosa cells: synthesis of hydroxyeicosatetraenoic acids and epoxyeicosatrienoic acids

Metabolites of arachidonic acid have been implicated in the regulation of aldosterone release. To form a basis for further investigations in this area, the present study has isolated and identified the metabolites formed from exogenous arachidonic acid by adrenal zona glomerulosa cells and characterized the effects of several inhibitors on the synthesis of these eicosanoids. Rat adrenal glomerulosa cells metabolized exogenous [14C]arachidonic acid to products comigrating with the prostaglandins (PGs), hydroxyeicosatatraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs). The metabolites were found in the cells and the incubation media; however, none of the metabolites were found esterified to cellular lipids. The major metabolites were identified as 6-keto PGF1 alpha, PGE2, PGF2 alpha, PGD2, 12(S)-HETE, 15(S)-HETE, 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET. The identities of the HETEs and EETs were confirmed by gas chromatography/mass spectrometry. There was no evidence for the synthesis of leukotrienes. The cyclooxygenase inhibitor, indomethacin, the lipoxygenase inhibitors, nordihydroguaiaretic acid, baicalein and AA861, and the combined cyclooxygenase/lipoxygenase inhibitors, BW755C and eicosatetrayenoic acid, inhibited the formation of the [14C]PGs, the [14C]HETEs, and the [14C]EETs. Metyrapone and clotrimazole, inhibitors of cytochrome P450, increased the synthesis of [14C]PGs and [14C]HETEs and reduced the synthesis of [14C] EETs. Superoxide dismutase did not alter arachidonic acid metabolism. In contrast, arachidonic acid metabolism was increased in cells pretreated with catalase. These data indicate that adrenal glomerulosa cells metabolize exogenous arachidonic acid to a number of oxygenated metabolites including PGs, HETEs, and EETs. From studies with inhibitors, the EETs appear to be synthesized by a cytochrome P450 epoxygenase and the HETEs by lipoxygenases.     

Incorporation of platelet and leukocyte lipoxygenase metabolites by cultured vascular cells

When arachidonic acid metabolism is studied during platelet-endothelial interactions in vitro, the predominant cyclooxygenase end products of each cell type (thromboxane B2 and 6-keto-prostaglandin-F1 alpha, respectively) are essentially completely recovered in the cell-free supernatants of these reactions. In contrast, 50% of 12-hydroxy- 5,8,10,14-eicosatetraenoic acid (12-HETE), the major lipoxygenase metabolite from platelets, is released into the cell-free supernatant. In investigating the basis of this observation, we have found that platelet lipoxygenase metabolites were generated to the same extent during these coincubations but became rapidly incorporated into the endothelial cells. The endothelial cell-associated 12-HETE was present not only as free fatty acid, but was also incorporated into cellular phospholipids and triglycerides. When purified 3H-12-HETE, 3H-5-HETE (the major hydroxy acid lipoxygenase product of leukocytes), and 3H- arachidonic acid (the common precursor of these metabolites) were individually incubated with suspensions of cultured bovine aortic endothelial cells or smooth muscle cells, different patterns of intracellular lipid distribution were found. In endothelial cells, 12- HETE was incorporated equally into phospholipids and triglycerides, whereas 5-HETE was incorporated preferentially into triglycerides, and arachidonic acid was incorporated into phospholipids. In smooth muscle cells, both 12-HETE and 5-HETE showed more extensive incorporation into triglycerides. The rapid and characteristic incorporation and esterification of platelet and leukocyte monohydroxy fatty acid lipoxygenase products by endothelial and smooth muscle cells suggests a possible physiologic role for these processes in regulating vascular function.     

Platelet modulation of polymorphonuclear leukocyte shear induced aggregation

A cone and plate viscometer and Coulter Counter were used to study platelet modulation of polymorphonuclear leukocyte (PMNL) aggregation caused by controlled shear stress. As an index of aggregation, the large-particle percentage (LPP) was calculated. This represents the ratio of aggregated cell count to total cell count. PMNL suspensions in buffer (1.0 X 10(7) cells per milliliter, final concentration) did not show any aggregate formation at shear stresses below 150 dynes/cm2 for one minute exposure time (LPP less than 3%). However, there was PMNL aggregation in mixed PMNL and platelet-rich plasma suspensions in this shear stress range. Supernatant plasma from sheared platelets initiated PMNL aggregation at moderate shear stress (150 dynes/cm2 for one minute; LPP, 20.3% +/- 2.5%). In contrast, platelet release factors, such as adenosine diphosphate (2 mumol/L) and serotonin (2 mumol/L) did not cause PMNL aggregation (LPP, 2.9% +/- 1.2% and 3.3% +/- 0.8%, respectively). The use of a cyclo-oxygenase inhibitor (acetylsalicylic acid, 50 mumol/L) did not suppress the aggregation of PMNLs after shear (LPP, 20.1% +/- 2.4%). However, preincubation with nordihydroguaiaretic acid (10 mumol/L), an inhibitor of C-5 and C-12 lipoxygenase, and 6,9- deepoxy-6,9-(phenylimino)-6,8-prostaglandin I1 (U-60257, 10 mumol/L), an inhibitor of C-5 lipoxygenase in human leukocytes, suppressed this aggregation (LPP, 9.1% +/- 2.5% and 10.4% +/- 3.2%, respectively). Also, the formation of lipoxygenase products (5-HETE, 12-HETE, 15-HETE, and LTB4) activated by shear stress was documented by reversed phase- high-performance liquid chromatography (RP-HPLC). These data support the possibility of a cooperation between platelets and leukocytes in shear-induced PMNL aggregation that is dependent on C-12 or C-5 lipoxygenase activity, or both.     

The effect of thrombin on the uptake and transformation of arachidonic acid by human platelets

Washed human platelets take up arachidonic acid from plasma and incorporate the fatty acid into the major classes of complex lipids. Thrombin impairs net incorporation. It activates endogenous phospholipases which liberate arachidonic acid from phospholipids. As a consequence of thrombin induced aggregation platelets release arachidonic acid intermediates formed by the action of platelet fatty acid cyclooxygenase and by platelet fatty acid lipoxygenase. Cyclooxygenase, but not lipoxygenase, is inhibited by aspirin and indomethicin. Analysis of the pathways of arachidonic acid metabolism may furnish new insight into platelet function and into disorders of primary hemostasis.     

Transmembrane signals mediating neural peptide secretion: role of protein kinase C activators and arachidonic acid metabolites in luteinizing hormone-releasing hormone secretion

The present experiments were designed to determine the effects of different activators of protein kinase C on the secretion of LHRH from median eminence nerve terminals incubated in vitro. The release of prostaglandin E2 (PGE2), a metabolite of arachidonic acid intimately involved in the secretion of LHRH, was also evaluated. Synthetic diacylglycerol [1,2-didecanoylglycerol (DiC10)] significantly enhanced PGE2 release in a concentration-dependent manner. Blockade of phospholipase A2 (PLA2) activity nullified this effect. LHRH release, on the other hand, was not increased by DiC10. However, in the presence of a lipoxygenase inhibitor, DiC10 produced a concentration-related increase in LHRH release, which paralleled that in PGE2. Phospholipase C (PLC) increased both PGE2 and LHRH secretion. Again, blockade of the lipoxygenase pathway enhanced the release of LHRH by PLC without affecting the stimulated secretion of PGE2. A phorbol ester, phorbol 12,13-dibutyrate (PDBu), markedly increased LHRH secretion while inducing a modest increase in PGE2 release. Both effects of PDBu were unaffected by lipoxygenase inhibition. DiC10, PDBu, and PLC significantly augmented LHRH secretion from tissues in which metabolism of arachidonic acid had been prevented by inhibition of both cyclooxygenase and lipoxygenase pathways, suggesting that activation of protein kinase C, independent of PLA2 activation, can lead to the secretion of this neural peptide. Some lipoxygenase metabolites had either no effect on [5- and 15-hydroxyeicosatetraenoic (5- and 15-HETE)] or induced a marginal stimulation of (12-HETE) LHRH release. At certain concentrations, 12-HETE enhanced the stimulatory effect of the phorbol ester on LHRH release. Our results suggest that activation of protein kinase C can stimulate LHRH secretion from nerve terminals in vitro and, further, that diacylglycerol may represent an important intracellular messenger participating in the events leading to LHRH secretion. In addition, stimulation with DiC10 and PLC uncovered inhibitory [unknown arachidonic acid metabolite(s) via lipoxygenase] and stimulatory (PGE2 via cyclooxygenase) pathways through with arachidonic acid metabolites may participate in the intracellular transduction of signals modulating neural peptide secretion.     

Poxvirus-induced alteration of arachidonate metabolism.

Recent evidence suggests that orthopoxviruses have an obligate requirement for arachidonic acid metabolites during replication in vivo and in vitro. Our report indicates that a virus family (Poxviridae) possesses multiple genes that function to regulate arachidonate metabolism. Analyses of BS-C-1 cells infected with cowpox virus or vaccinia virus detected enhanced arachidonate product formation from both the cyclooxygenase (specifically prostaglandins E2 and F2 alpha) and lipoxygenase (specifically 15-hydroxyeicosatetraenoic acid and 12-hydroxyeicosatetraenoic acid) pathways. In contrast, human parainfluenza type 3 or herpes simplex virus type 1 infections did not increase arachidonate metabolism. Results were consistent with a virus early-gene product either directly mediating or inducing a host factor that mediated the up-regulation of arachidonate metabolism, although vaccinia growth factor was not responsible. In addition, the cowpox virus 38-kDa protein-encoding gene, which is associated with inhibition of an inflammatory response, correlated with inhibition of formation of a product biochemically characteristic of (14R,15S)-dihydroxyeicosatetraenoic acid. We propose that orthopoxvirus-induced up-regulation of arachidonic acid metabolism during infection renders the infected cells susceptible to generation of inflammatory mediators from both the cyclooxygenase and the lipoxygenase pathways, and poxviruses, therefore, possess at least one gene (38K) that can alter the lipoxygenase-metabolite spectrum.     

12(S)-HETE plays a role as a mediator of expression of platelet CD62 (P-selectin)

The role of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE), an abundant lipoxygenase product from arachidonic acid in platelets, remains unknown. We investigated and characterized the role of 12(S)-HETE in platelet activation. 12(S)-HETE production and CD62 expression were increased by stimulation with thrombin at 0.03 U/ml or higher, while TXB2 synthesis was increased by thrombin at 0.1 U/ml or higher. The platelet 12(S)-HETE production was increased 10 s after stimulation and this was earlier than CD62 expression. The expression of CD62 was inhibited by the lipoxygenase (LOX) inhibitors quercetin and nordihydroguaiaretic acid but was not affected by the cyclooxygenase (COX) inhibitors aspirin and indomethacin. Exogenously added 12(S)-HPETE and 12(S)-HETE enhanced CD62 expression, but other HETEs did not. Consequently, 12-LOX products play a role in the expression of CD62 and could be a second messenger for platelet activation.     

12(S)-HETE plays a role as a mediator of expression of platelet CD62 (P-selectin)

The role of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE), an abundant lipoxygenase product from arachidonic acid in platelets, remains unknown. We investigated and characterized the role of 12(S)-HETE in platelet activation. 12(S)-HETE production and CD62 expression were increased by stimulation with thrombin at 0.03 U/ml or higher, while TXB2 synthesis was increased by thrombin at 0.1 U/ml or higher. The platelet 12(S)-HETE production was increased 10 s after stimulation and this was earlier than CD62 expression. The expression of CD62 was inhibited by the lipoxygenase (LOX) inhibitors quercetin and nordihydroguaiaretic acid but was not affected by the cyclooxygenase (COX) inhibitors aspirin and indomethacin. Exogenously added 12(S)-HPETE and 12(S)-HETE enhanced CD62 expression, but other HETEs did not. Consequently, 12-LOX products play a role in the expression of CD62 and could be a second messenger for platelet activation.     

Modulation of arachidonic acid metabolism by olsalazine and other aminosalicylates in leukocytes.

We investigated the action of the new aminosalicylate olsalazine (disodium azodisalicylate) on arachidonic acid metabolism in comparison with 5-aminosalicylic acid (5-ASA) and sulphasalazine (SASP) by in vitro incubation of cellular homogenates from human polymorphonuclear (PMNL) and mononuclear (MNL) leukocytes with 14C-labelled arachidonic acid. Olsalazine reduced the synthesis of leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoic acid (5-HETE), 11-HETE, 12-HETE, and 15-HETE in PMNL and MNL slightly less than SASP. 5-ASA was significantly less inhibitory than olsalazine and SASP on the formation of lipoxygenase products in PMNL and on LTB4 synthesis in MNL. In contrast, in MNL the formation of 5-HETE was unaffected, and the production of 11-HETE, 12-HETE, and 15-HETE was even slightly activated by 5-ASA. Total prostaglandin synthesis was dose-dependently reduced by the aminosalicylates (SASP greater than olsalazine greater than 5-ASA), but only SASP markedly altered the prostaglandin (PG) profile, with an increase in PGE2 and PGF2 alpha at the expense of other cyclooxygenase products. It may be concluded that olsalazine resembled SASP with regard to the inhibition of the lipoxygenase but had effects intermediate between the other salicylates on cyclooxygenase. Furthermore, the alteration of the prostaglandin profile by SASP points to an overlying cofactor effect of this drug.     

Effect of stress and the adaptogen cucurbitacin R diglycoside on arachidonic acid metabolism

Rat leukocytic lipoxygenase activity is decreased in stress. The production of 12-hydroxy-5z, 8z, 10E-heptadecatrienic acid (12-HHT) (the product of cyclooxygenase metabolism of arachidonic acid-AA) is increased. Cucurbitacin R diglucoside (CRD), an adaptogen, raising working capacity and corticosteroid secretion, produces a similar effect on leukocytes. Preliminary injection of CRD to animals prevents changes caused by stress, which indicates a CRD adaptogenic effect on the body.     

Leukotriene C: a slow-reacting substance from murine mastocytoma cells.

Murine mastocytoma cells treated with calcium ionophore A23187 produced a slow-reacting substance (SRS) that caused guinea pig ileum to contract. The response was reversed by the SRS antagonist FPL 55712. On the basis of isotope incorporation experiments, spectroscopy, and chemical degradations, the SRS was identified as a cysteine-containing derivative of 5-hydroxy-7,9,11,14-icosatetraenoic acid. This amino acid was attached in thioether linkage at C-6. The SRS is structurally related to previously identified epoxy and dihydroxy metabolites of arachidonic acid in leukocytes. A common feature is the presence of a conjugated triene, and the name "leukotriene" has been introduced to designate these compounds. Leukotriene A (5,6-epoxy-7,9,11,14-icosatetraenoic acid) is an intermediate in the formation of leukotriene B (5,12-dihydroxy-6,8,10,14-icosatetraenoic acid) and is proposed to be a precursor also of leukotriene C, which is the SRS identified here.     

Role of arachidonate lipoxygenase and cyclooxygenase products in insulin and glucagon secretion from rat pancreatic islets

Rat pancreatic islets incubated in nutrient medium were used to study the role of endogenous arachidonic acid metabolism in pancreatic hormone secretion. Both glucose and fetal calf serum stimulated radioimmunoassayable PGE₂ production and insulin secretion from islets. These effects were abolished by the phospholipase inhibitor p-bromophenacyl bromide or by concurrent inhibition of cyclooxygenase and lipoxygenase by flurbiprofen plus nordihydroguaiaretic acid (NDGA), respectively. Bromophenacyl bromide also inhibited glucagon secretion. When used alone, flurbiprofen caused a significant enhancement of glucose-induced insulin secretion that was attributed to reactive stimulation of lipoxygenase-product formation rather than to selective cyclooxygenase inhibition. NDGA given alone in the presence of stimulatory concentrations of glucose suppressed the normal eight-fold rise in insulin secretion, but caused a marked enhancement in glucagon secretion that could be overcome by simultaneous inclusion of flurbiprofen. We concluded that: (1) Increased metabolism of arachidonic acid in pancreatic islets amplifies the secretion of insulin and glucagon. (2) The lipoxygenase as well as the cyclooxygenase pathways of arachidonate metabolism participate in the amplification of insulin secretion. (3) The observations made in this study are inconclusive with respect to the involvement of the lipoxygenase and cyclooxygenase pathways in glucagon secretion; an inhibitory role for lipoxygenase pathway products is suggested.     

Precursor role of arachidonic acid in release of slow reacting substance from rat basophilic leukemia cells.

The release of slow reacting substance (SRS) from rat basophilic leukemia cells (RBL-1) by the ionophore A23187 (5-10 mug/ml) was stimulated 5-fold by arachidonate and inhibited 78% by 5,8,11,14-eicosatetraynoate (an inhibitor of both fatty acid cyclooxygenase and lipoxygenase). Linoleic acid and linolenic acid both inhibited SRS formation, whereas indomethacin (a cyclooxygenase inhibitor) had no effect. Radiolabel from [14C]- or [3H]arachidonate was incorporated into SRS as indicated by comigration of radioactivity and bioreactivity in several chromatographic systems after purification to apparent radiochemical homogeneity. The radiolabeled SRS was clearly separated chromatographically from other known arachidonate metabolites. Thus, SRS appears to be a previously undescribed product of arachidonic acid metabolism, probably formed through the lipoxygenase pathway. The ability to prepare purified, biosynthetically labeled, SRS should be of considerable help in further studies of its structure, biologic function, and catabolism.     

Lipoxins: novel series of biologically active compounds formed from arachidonic acid in human leukocytes.

Trihydroxytetraenes, a novel series of oxygenated derivatives formed from arachidonic acid in human leukocytes, were recently isolated [Serhan, C. N., Hamberg, M. & Samuelsson, B. (1984) Biochem. Biophys. Res. Commun. 118, 943-949]. The structure of the major compound was established--i.e., 5,6,15L-trihydroxy-7,9,11,13-icosatetraenoic acid. The present study reports the structure of a second member of the trihydroxytetraene series of compounds--i.e., 5D,14,15L-trihydroxy-6,8,10,12-icosatetraenoic acid. When added to human neutrophils, 5,6,15L-trihydroxy-7,9,11,13-icosatetraenoic acid stimulated superoxide anion generation and degranulation at submicromolar concentrations without provoking a substantial aggregation response. With respect to superoxide anion generation, 5,6,15L-trihydroxy-7,9,11,13-icosatetraenoic acid proved to be as potent as leukotriene B4. In contrast, the compound was approximately 2 orders of magnitude less potent than either leukotriene B4 or fMet-Leu-Phe at provoking degranulation. The results indicate that interaction(s) between the 5- and 15-lipoxygenase pathways of human leukocytes leads to formation of a new series of oxygenated derivatives of arachidonic acid that may be involved in regulating specific cellular responses. The trivial names lipoxin A (5,6,15L-trihydroxy-7,9,11,13-icosatetraenoic acid) and lipoxin B (5D,14,15L-trihydroxy-6,8,10,12-icosatetraenoic acid) are proposed for the new compounds.     

Deficient induction of leukotriene synthesis in human neutrophils by lipoxygenase-deficient platelets

The effect of human platelets with deficient lipoxygenase activities on leukotriene B4 (LTB4) synthesis by neutrophils was studied. When arachidonic acid (AA) metabolites obtained from the incubation of washed normal neutrophils and platelets with N- formylmethionylleucylphenylalanine (FMLP), cytochalasin B, and AA were analyzed by reversed-phase high-performance liquid chromatography, the synthesis of 5-lipoxygenase products, including LTB4, was remarkably stimulated by platelets, with their maximal effect at a ratio of platelets to neutrophils of 15:1. However, the use of lipoxygenase- deficient platelets obtained from four patients with myeloproliferative disorders instead of normal platelets showed the deficient production of 5-lipoxygenase-derived products, whereas platelets with normal lipoxygenase activities obtained from MPD patients stimulated the 5- lipoxygenase pathway similarly to the way in which normal platelets did. The addition of 12-hydroperoxyeicosatetraenoic acid (12-HPETE), a labile AA metabolite via the platelet lipoxygenase pathway, could activate the 5-lipoxygenase pathway in neutrophils incubated with FMLP, cytochalasin B and AA, but its stable end product, 12- hydroxyeicosatetraenoic acid, could not. Thus, it is suggested that lipoxygenase-deficient platelets did not sufficiently stimulate LTB4 synthesis during platelet-neutrophil interactions because of defective formation of 12-HPETE. This altered interaction between platelets and neutrophils through the lipoxygenase pathway might result in deficient responses at sites of thrombosis or inflammation in patients with deficient platelet lipoxygenase activities.     

Arachidonic acid metabolism in polymorphonuclear leukocytes: unstable intermediate in formation of dihydroxy acids.

An unstable intermediate was detected in the transformation of arachidonic acid into 5,6-dihydroxyicosatetraenoic acids (two isomers) and 5,12-dihydroxyicosatetraenoic acids (three isomers) in rabbit peritoneal (glycogen-induced) polymorphonuclear leukocytes. Addition of 10 vol of methanol, ethanol, or ethylene glycol to short-term incubations (30-45 sec) led to the formation of the corresponding 12-O-alkyl derivatives of the 5,12-dihydroxy acids. The time for 50% disappearance of the intermediate (37 degrees C), as measured by formation of 5-hydroxy-12-O-methylicosatetraenoic acids (two isomers) upon trapping with methanol, was about 1 min in live cell preparations (pH 7.4) and about 4 min in water/acetone (1:1), pH 7.4. At pH 6.0 or below, the hydrolysis of the intermediate was too rapid to be measured by the method employed. Data supporting both enzymatic and nonenzymatic hydrolysis of the intermediate into dihydroxy acids are presented. Incubation of the cells with arachidonic acid under an atmosphere of 18O2 led to incorporation of 18O into the 5,6-dihydroxy acids and 5,12-dihydroxy acids only at C-5. The 5-hydroxyicosatetraenoic acid was also labeled at C-5. Considering the chemical reactivity of the intermediate and the structures of the derivatives obtained, it is proposed that the intermediate is 5(6)-oxido-7,9,11,14-icosatetraenoic acid.     

Norepinephrine and thyrotropin stimulation of iodide efflux in FRTL-5 thyroid cells involves metabolites of arachidonic acid and is associated with the iodination of thyroglobulin

Ca2+-dependent and TSH-, norepinephrine (NE)-, and A23187-induced iodide (I-) efflux from FRTL-5 rat thyroid cells is inhibited by quinacrine and trifluoroperazine, agents that inhibit phospholipase A2 activity. Furthermore, I- efflux can be stimulated by an activator of phospholipase A2 activity, melittin. Phospholipase A2 action releases arachidonic acid from phospholipids; arachidonic acid enhances I- efflux in FRTL-5 cells. Inhibitors of arachidonic acid metabolism via the lipoxygenase pathway, 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid, and via the cytochrome P450-linked epoxygenase pathway, piperonyl butoxide and 2-diethylaminoethyl-2,2-diphenyl valerate, but not an inhibitor of the cyclooxygenase pathway, indomethacin, can inhibit TSH-, NE-, and A23187-induced I- efflux. TSH, NE, and arachidonic acid stimulation of I- efflux in FRTL-5 cells is associated with increased iodination of thyroglobulin, which is blocked by 10 microM 5,8,11,14-eicosatetraynoic acid and 50 microM piperonyl butoxide. The data thus suggest that TSH- and NE-induced I- efflux from FRTL-5 thyroid cells involves lipoxygenase and/or epoxygenase metabolites of arachidonic acid, released from phospholipids upon Ca2+-dependent activation of phospholipase A2. Since this process is associated with the iodination of thyroglobulin, TSH- and NE-induced I- efflux in FRTL-5 cells may represent the transport of I- from the cell into the follicular lumen in vivo.