9,11-Iminoepoxyprosta-5,13-dienoic acid is a selective thromboxane A2 synthetase inhibitor.

9,11-Iminoepoxyprosta-5,13-dienoic acid inhibits the thromboxane A2 synthetase in platelet and lung microsomal enzyme preparations and in intact platelets. It does not inhibit the protaglandin I2 synthetase in aorta or lung microsomes and intact Balb 3T3 fibroblasts. In lung microsomes, which contain both enzymes, 9,11-iminoepoxyprosta-5,13-dienoic acid inhibits only thromboxane A2 formation and augments prostaglandin I2 formation. This inhibitor is more selective than other reported prostaglandin endoperoxide analogs which inhibit the platelet thromboxane synthetase.     

Nicotinic acid inhibits thromboxane synthesis in platelets

The formation of thromboxane A₂ by phospholipase A₂ in rat platelets is inhibited by nicotinic acid. The synthesis of PGE₂ and PGF_2α is increased.Nicotinic acid inhibits collagen-induced aggregation in rat platelets.     

A comparison of imidazole and 9,11-azoprosta-5,13-dienoic acid Two selective thromboxane synthetase inhibitors

Two selective thromboxane A₂ synthetase inhibitors, imidazole and 9,11-azoprosta-5,13-dienoic acid (azo analog I) were compared to determine their effects on the quantitative formation of thromboxane B₂ and prostaglandin E₂ accompanying human platelet aggregation. Azo analog I was at least 200 times more potent, on a molar basis, than imidazole in suppressing thromboxane B₂ formation in either platelet-rich plasma or washed platelet suspensions aggregated with arachidonic acid or prostaglandin H₂. The inhibitors differed in their effect on the aggregation response itself. Azo analog I selectively suppressed thromboxane A₂ formation with an accompanying, parallel, suppression of the platelet aggregation.Imidazole selectively suppressed thromboxane A₂ formation, but only suppressed the accompanying aggregation in platelet rich plasma, and not washed platelet suspensions. The results indicate that azo analog I functions by competitive inhibition of prostaglandin H₂ on the thromboxane synthetase, and that imidazole, while it suppresses thromboxane A₂ formation, may have an associated agonist activity that enhances platelet aggregation. The data presented support this hypothesis, and they emphasize the importance of thromboxane A₂ in arachidonate mediated platelet aggregation.     

A comparison of imidazole and 9,11-azoprosta-5,13-dienoic acid. Two selective thromboxane synthetase inhibitors

Two selective thromboxane A2 synthetase inhibitors, imidazole and 9,11-azoprosta-5,13-dienoic acid (azo analog I) were compared to determine their effects on the quantitative formation of thromboxane B2 and prostaglandin E2 accompanying human platelet aggregation. Azo analog I was at least 200 times more potent, on a molar basis, than imidazole in suppressing thromboxane B2 formation in either platelet-rich plasma or washed platelet suspensions aggregated with arachidonic acid or prostaglandin H2. The inhibitors differed in their effect on the aggregation response itself. Azo analog I selectively suppressed thromboxane A2 formation with an accompanying, parallel, suppression of the platelet aggregation. Imidazole selectively suppressed thromboxane A2 formation, but only suppressed the accompanying aggregation in platelet rich plasma, and not washed platelet suspensions. The results indicate that azo analog I functions by competitive inhibition of prostaglandin H2 on the thromboxane synthetase, and that imidazole, while it suppresses thromboxane A2 formation, may have an associated agonist activity that enhances platelet aggregation. The data presented support this hypothesis, and they emphasize the importance of thromboxane A2 in arachidonate mediated platelet aggregation.     

12-Hydroxy-5Z, 8Z, 10E, 14Z,eicosatetraenoic acid (12-HETE) stimulates cAMP production in normal human fibroblasts

We report here that the 12-lipoxygenase metabolite of arachidonic acid, 12-hydroxy-5Z, 8Z, 10E, 14Z, eicosatetraenoic acid (12-HETE), stimulates cAMP production in human fibroblasts among various cultured cell lines tested. Although 12-HETE seemed to stimulate the phospholipase C (PLC)-protein kinase C (PKC) system, inhibitors against PLC and PKC did not reduce the cAMP production induced by 12-HETE, indicating that the activation of PLC-PKC system is not positively coupled with the stimulation of cAMP production. On the other hand, the cAMP production induced by 12-HETE was dependent on the Ca²⁺/calmodulin system in the cells. The results suggest that 12-HETE specifically stimulates Ca²⁺/calmodulin-dependent adenylyl cyclase to increase cAMP level in the fibroblasts. J Cell Physiol 178:63–68, 1999. © 1999 Wiley-Liss, Inc.     

Effect of 13-hydroperoxy-9,11-octadecadienoic acid (13-hpode) on arachidonic acid metabolism in rabbit platelets

• 1.1. The effect of 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE) on the formation of thromboxane (TX) B₂, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from exogenous arachidonic acid in washed rabbit platelets was examined.
• 2.2. 13-HPODE inhibited TXB₂ and HHT formation without affecting 12-HETE production.
• 3.3. 13-Hydroxy-9,11-octadecadienoic acid which was produced rapidly from 13-HPODE, did not suppress the formation of TXB₂ and HHT, indicating the requirement of the hydroperoxy moiety for the inhibitory effect of 13-HPODE on TXB₂ and HHT formation.
• 4.4. Experiments utilizing mannitol and dimethyl sulfoxide (hydroxy radical scavengers) revealed that the action of 13-HPODE is not due to hydroxy radicals which are expected to be formed from 13-HPODE.
• 5.5. These results suggest that 13-HPODE is a selective inhibitor of platelet cyclo-oxygenase and may have functional effects within platelets.

     

Lanthanum stimulates the accumulation of cyclic AMP and inhibits secretion and thromboxane B2 formation in human platelets

La3+ was found to inhibit the secretion of 5-hydroxytryptamine and the production of thromboxane B2 by washed platelets exposed to collagen or thrombin. In addition, La3+ inhibited secretion in response to sodium arachidonate, although the conversion of arachidonate to thromboxane B2 was not affected. La3+ was also found to enhance the accumulation of cyclic AMP under basal conditions and in response to prostaglandin E1, in washed platelets. The inhibition of cyclic AMP accumulation by ADP was prevented by La3+, suggesting that the effect of ADP on cyclic AMP metabolism was dependent upon the presence or flux of calcium at the platelet membrane. La3+ inhibited the activity of adenylate cyclase in platelet lysates both in response to prostaglandin E1 and to F-, indicating a possible effect at the catalytic subunit of the enzyme. None of the observed effects of La3+ could be reversed by the addition of Ca2+ up to 10 mM. The stimulation of cyclic AMP production by La3+ may largely explain the inhibitory effect of La3+ upon platelet secretion and thromboxane B2 production. These results also suggest that Ca2+ localised at the platelet plasma membrane may be important in the regulation of cyclic AMP metabolism.     

Anti-proliferative lichen compounds with inhibitory activity on 12(S)-HETE production in human platelets

Several lichen compounds, i.e. lobaric acid (1), a β-orcinol depsidone from Stereocaulon alpinum L., (+)-protolichesterinic acid (2), an aliphatic -methylene-γ-lactone from Cetraria islandica Laur. (Parmeliaceae), (+)-usnic acid (3), a dibenzofuran from Cladonia arbuscula (Wallr.) Rabenh. (Cladoniaceae), parietin (4), an anthraquinone from Xanthoria elegans (Link) Th. Fr. (Calaplacaceae) and baeomycesic acid (5), a β-orcinol depside isolated from Thamnolia vermicularis (Sw.) Schaer. var. subuliformis (Ehrh.) Schaer. were tested for inhibitory activity on platelet-type 12(S)-lipoxygenase using a cell-based in vitro system in human platelets. Lobaric acid (1) and (+)-protolichesterinic acid (2) proved to be pronounced inhibitors of platelet-type 12(S)-lipoxygenase, whereas baeomycesic acid (5) showed only weak activity (inhibitory activity at a concentration of 100 μg/ml: 1 93.4±6.62%, 2 98,5±1.19%, 5 14.7±2.76%). Usnic acid (3) and parietin (4) were not active at this concentration. 1 and 2 showed a clear dose–response relationship in the range of 3.33–100 μg/ml. According to the calculated IC₅₀ values the highest inhibitory activity was observed for the depsidone 1 (IC₅₀=28.5 μM) followed by 2 (IC₅₀=77.0 μM). The activity of 1 was comparable to that of the flavone baicalein, which is known as a selective 12(S)-lipoxygenase inhibitor (IC₅₀=24.6 μM).     

12-Hydroxyeicosatetraenoic acid (12-HETE) induces heat shock proteins in human leukocytes

12-Hydroxyeicosatetraenoic acid (12-HETE) induces the expression of individual heat shock proteins in human leukocytes (lymphocytes, monocytes, basophil granulocytes; LMBs). Metabolic radiolabeling of LMBs revealed that exogenous 12-HETE (20 ng) led to the expression of a 65- and 83kDa protein. Immunoreactivity towards the 65kDa protein was commonly detected. In contrast, after heat shock treatment and predominantly after incubation with 12-HETE significant immunoreactivity (anti-hsp72) was detected in the lower molecular weight range whereas immunoreactivity (anti-hsp90, AC88) was only observed after heat shock treatment of LMBs.     

12-O-Tetradecanoylphorbol 13-acetate stimulates inositol lipid phosphorylation in intact human platelets

The phorbol esters are among the most potent tumor promoters. On addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) to isolated human platelets prelabelled with [32P]orthophosphate we found a rapid increase in 32P incorporation into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. In view of similar findings with cells infected with the oncogene Rous sarcoma virus, it is suggested that inositol lipid phosphorylation might be a key event in the molecular action of phorbol esters.     

Cyclic AMP regulation of endothelial cell triacylglycerol turnover, 13-hydroxyoctadecadienoic acid (13-HODE) synthesis and endothelial cell thrombogenicity

The 15-omega-lipoxygenase enzyme in endothelial cells metabolizes endogenous linoleic acid (18:2) into 13-hydroxyoctadecadienoic acid (13-HODE) under basal conditions, i.e., in unstimulated endothelial cells. 13-HODE is thought to regulate the non-adhesivity of the endothelium, contributing to vessel wall/blood cell biocompatibility. We performed experiments, therefore, to determine the relationship between basal levels of cAMP, 13-HODE synthesis, and platelet/endothelial cell adhesion. We found that 13-HODE synthesis increased with elevated cAMP levels and that the elevated 13-HODE levels correlated with increased 18:2 turnover in the triacylglycerol pool. In contrast, neither 18:2 nor arachidonic acid (20:4) turnover in the phospholipid nor prostacyclin (PGI2) production were changed with elevated cAMP levels. Platelet/endothelial cell adhesion was inversely proportional to 13-HODE synthesis. We conclude that intracellular 13-HODE influences platelet/vessel wall interactions, is synthesized from 18:2 released from the endogenous triacylglycerol pool, and that this pathway is modulated by intracellular cAMP levels.     

Lanthanum stimulates the accumulation of cyclic AMP and inhibits secretion and thromboxane B2 formation in human platelets

La³⁺ was found to inhibit the secretion of 5-hydroxytryptamine and the production of thromboxane B₂ by washed platelets exposed to collagen or thrombin. In addition, La³⁺ inhibited secretion in response to sodium arachidonate, although the conversion of arachidonate to thromboxane B₂ was not affected.La³⁺ was also found to enhance the accumulation of cyclic AMP under basal conditions and in response to prostaglandin E₁, in washed platelets. The inhibition of cyclic AMP accumulation by ADP was prevented by La³⁺, suggesting that the effect of ADP on cyclic AMP metabolism was dependent upon the presence or flux of calcium at the platelet membrane.La³⁺ inhibited the activity of adenylate cyclase in platelet lysates both in response to prostaglandin E₁ and to F^?, indicating a possible effect at the catalytic subunit of the enzyme. None of the observed effects of La³⁺ could be reversed by the addition of Ca²⁺ up to 10 mM. The stimulation of cyclic AMP production by La³⁺ may largely explain the inhibitory effect of La³⁺ upon platelet secretion and thromboxane B₂ production. These results also suggest that Ca²⁺ localised at the platelet plasma membrane may be important in the regulation of cyclic AMP metabolism.     

Cyclic AMP inhibits synthesis of prostaglandin endoperoxide (PGG2) in human platelets.

Dibutyryl-cAMP but not dibutyryl-cGMP inhibited platelet aggregation and release of ¹⁴C-serotonin and ADP when induced by collagen and arachidonate but not when induced by the endoperoxide PGG₂^* (TXB₂) induced by addition of collagen to platelet rich plasma (PRP) was decreased by dibutyryl-cAMP and agents known to increase the concentration of cAMP (PGE₁, PGD₂, theophylline and acetyl choline).PGE₂ in concentrations known to decrease cAMP levels increased the formation of TXB₂ whereas concentrations of PGE₂ known to increase cAMP levels decreased the amount of TXB₂ formed. That this was due to an effect on the cyclooxygenase was indicated by inhibition of the transformation of arachidonic acid by DB-cAMP and by high concentrations of PGE₂. Additional support for regulation of the cyclo-oxygenase by cAMP and its relevance to platelet aggregation was obtained by demonstrating stimulation of PGG₂ induced aggregation by low concentrations of PGE₂ and the absence of this effect in the presence of a cyclo-oxygenase inhibitor.     

Lipoxygenase-mediated metabolism of storage lipids in germinating sunflower cotyledons and beta-oxidation of (9Z,11E,13S)-13-hydroxy-octadeca-9,11-dienoic acid by the cotyledonary glyoxysomes

During the early stages of germination, a lipid-body lipoxygenase is expressed in the cotyledons of sunflowers (Helianthus annuus L.). In order to obtain evidence for the in vivo activity of this enzyme during germination, we analyzed the lipoxygenase-dependent metabolism of polyunsaturated fatty acids esterified in the storage lipids. For this purpose, lipid bodies were isolated from etiolated sunflower cotyledons at different stages of germination, and the storage triacylglycerols were analyzed for oxygenated derivatives. During the time course of germination the amount of oxygenated storage lipids was strongly augmented, and we detected triacylglycerols containing one, two or three residues of (9Z,11E,13S)-13-hydro(pero)xy-octadeca-9,11-dienoic acid. Glyoxysomes from etiolated sunflower cotyledons converted (9Z,11E,13S)-13-hydroxy-octadeca-9,11-dienoic acid to (9Z,11E)-13-oxo-octadeca-9,11-dienoic acid via an NADH-dependent dehydrogenase reaction. Both oxygenated fatty acid derivatives were activated to the corresponding CoA esters and subsequently metabolized to compounds of shorter chain length. Cofactor requirement and formation of acetyl-CoA indicate degradation via -oxidation. However, -oxidation only proceeded for two consecutive cycles, leading to accumulation of a medium-chain metabolite carrying an oxo group at C-9, equivalent to C-13 of the parent (9Z,11E,13S)-13-hydroxy-octadeca-9,11-dienoic acid. Short-chain -oxidation intermediates were not detected during incubation. Similar results were obtained when 13-hydroxy octadecanoic acid was used as -oxidation substrate. On the other hand, the degradation of (9Z,11E)-octadeca-9,11-dienoic acid was accompanied by the appearance of short-chain -oxidation intermediates in the reaction mixture. The results suggest that the hydroxyl/oxo group at C-13 of lipoxygenase-derived fatty acids forms a barrier to continuous -oxidation by glyoxysomes.     

Inhaled PAF stimulates leukotriene and thromboxane A2 production in humans.

Platelet-activating factor (PAF) is a potent bronchoconstrictor in humans and has been implicated as an inflammatory mediator in asthma. This study was performed to evaluate whether PAF-induced bronchoconstriction in vivo could be mediated through the release of the bronchoconstrictor eicosanoids, thromboxane (Tx) A2 and the cysteinyl leukotrienes. Ten asthmatic subjects were studied on three occasions after bronchial challenges with aerosolized PAF, methacholine, or isotonic saline. PAF caused bronchoconstriction in all 10 subjects (mean maximal percent fall in specific airway conductance 48.2 +/- 4.6) and was matched by methacholine challenge. Saline caused no changes in specific airway conductance. Urinary leukotriene E4 was significantly elevated after inhaled PAF (366.0 +/- 66.9 ng/mmol creatinine, P less than 0.01) compared with methacholine (41.6 +/- 13.3) and saline (33.6 +/- 4.6). The major urinary TxA2 metabolite 2,3-dinor TxB2 was elevated after inhaled PAF (41.3 +/- 7.1 ng/mmol creatinine, P less than 0.01) compared with methacholine (14.0 +/- 2.7) and saline (17.1 +/- 3.9). Urinary 2,3-dinor 6-oxo-prostaglandin F1 alpha after PAF (22.2 +/- 1.4) was raised with respect to the methacholine challenge (13.9 +/- 1.8, P less than 0.02), although no significant increase was observed compared with the saline control (18.6 +/- 3.3). Inhaled PAF leads to the secondary generation of cysteinyl leukotrienes and TxA2, and it is possible that these mediate some of the acute effects of inhaled PAF in vivo.     

Binding of 13-HODE and 5-, 12- and 15-HETE to endothelial cells and subsequent platelet, neutrophil and tumor cell adhesion

Some studies report that endothelial cells preferentially take up the lipoxygenase-derived arachidonic acid metabolite, 5-hydroxyeicosatetraenoic acid (5-HETE), released from stimulated leukocytes (polymorphonuclear leukocytes, PMNs), whereas others report that endothelial cells preferentially take up 12-HETE released from platelets. The biological relevance of these observations, however, is unknown. Recently, we and others have found that, under basal conditions, endothelial cells, PMNs and tumor cells metabolize linoleic acid via the lipoxygenase enzyme to 13-hydroxyoctadecadienoic acid (13-HODE). We propose that endogenous levels of these metabolites regulate blood-vessel wall cell adhesion. In this study, we have measured (1) the relative binding of 5-, 12- and 15-HETE, and 13-HODE to endothelial cell monolayers, and (2) their effects on endothelial cell adhesivity with platelets, PMNs and tumor cells. There was a dose-related and specific binding of 5-[3H]HETE to endothelial cells but no binding of 12- or 15-HETE or 13-HODE. Platelet or PMN adhesion to endothelial cells was unaffected by the 5-HETE binding, but tumor cell adhesion was blocked by 40% (P less than 0.01). Interestingly, preincubation of endothelial cells with 13-HODE, 12-HETE or 15-HETE decreased platelet adhesion to endothelial cells (P less than 0.05), even though these metabolites did not bind to the endothelial cells. We conclude that 5-HETE preferentially binds to endothelial cells and interferes with a specific receptor for tumor cells, whereas the other metabolites neither bind to cells nor affect cell adhesion.     

Eicosapentaenoic acid and docosahexaenoic acid are antagonists at the thromboxane A2/prostaglandin H2 receptor in human platelets

The present study investigated the mechanism by which eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) inhibit platelet activation induced by thromboxane A2. DHA was found to be more potent than EPA in blocking platelet aggregation induced by the stable thromboxane A2 mimetic, U46619. Furthermore, this inhibition by DHA or EPA was competitive. Binding studies using 3H-U46619 demonstrated that both EPA and DHA interact with the platelet thromboxane receptor. The potency of the inhibition of binding corresponded with that seen for the inhibition of aggregation. These results suggest that thromboxane receptor antagonism may be an important mechanism by which EPA and DHA modulate platelet reactivity in vivo.     

13-(S)-hydroxyoctadecadienoic acid (13-HODE) incorporation and conversion to novel products by endothelial cells

13(S)-Hydroxy-[12,13-3H]octadecadienoic acid (13-HODE), a linoleic acid oxidation product that has vasoactive properties, was rapidly taken up by bovine aortic endothelial cells. Most of the 13-HODE was incorporated into phosphatidylcholine, and 80% was present in the sn -2 position. The amount of 13-HODE retained in the cells gradually decreased, and radiolabeled metabolites with shorter reverse-phase high-performance liquid chromatography retention times (RT) than 13-HODE accumulated in the extracellular fluid. The three major metabolites were identified by gas chromatography combined with mass spectrometry as 11-hydroxyhexadecadienoic acid (11-OH-16:2), 9-hydroxytetradecadienoic acid (9-OH-14:2), and 7-hydroxydodecadienoic acid (7-OH-12:2). Most of the radioactivity contained in the cell lipids remained as 13-HODE. However, some 11-OH-16:2 and several unidentified products with longer RT than 13-HODE were detected in the cell lipids. Normal human skin fibroblasts also converted 13-HODE to the three major chain-shortened metabolites, but Zellweger syndrome fibroblasts produced only a very small amount of 11-OH-16:2. Therefore, the chain-shortened products probably are formed primarily by peroxisomal beta-oxidation. These findings suggest that peroxisomal beta-oxidation may constitute a mechanism for the inactivation and removal of 13-HODE from the vascular wall. Because this is a gradual process, some 13-HODE that is initially incorporated remains in endothelial phospholipids, especially phosphatidylcholine. This may be the cause of some of the functional perturbations produced by 13-HODE in the vascular wall.     

Regulation of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) binding sites on human epidermal cells by interferon-gamma

We recently detected specific high-affinity binding sites for 12(S)-HETE, the main arachidonic acid metabolite in skin, on epidermal cells. The putative receptor is involved in keratinocyte chemotaxis toward 12(S)-HETE, which points to its participation in wound healing. In an effort to further characterize the 12(S)-HETE receptor, we investigated its regulation by various cytokines. Of the tested cytokines, only interferon (IFN)-gamma led to a massive induction of the 12(S)-HETE receptors. The effect was dose and time dependent and blocked by cycloheximide. The up-regulation of 12(S)-HETE receptors by IFN-gamma may represent an amplification mechanism of the assumed role of 12(S)-HETE in skin wound repair.