The appearance of phospholipase activity in the human macrophage-like cell line U937 during dimethyl sulfoxide induced differentiation

The human histiocyte cell line, U937, with monocyte characteristics, can be induced to differentiate into macrophage-like cells when exposed to growth medium containing 1.5% DMSO. Following three days of exposure, DMSO-treated but not control U937 cells can be stimulated to release endogenous arachidonic acid from their phospholipids. Maximum release of the unsaturated fatty acid occurs with 10 microM calcium ionophore in the presence but not in the absence of exogenously added calcium ion. In addition, DMSO-treated but not control U937 cells exhibit phospholipase activity when exposed to human IgG and then anti-human immunoglobulin. These data suggest that with respect to arachidonic acid metabolism U937 cells differentiate into functional macrophage-like cells when exposed to DMSO.     

Preferential utilization of endogenous arachidonate by cyclo-oxygenase in incubations of human platelets

Thromboxane B2 (TXB2) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) formed from the endogenous and exogenous arachidonate during human platelet incubation, was evaluated by selected ion monitoring (SIM). TXB2 formed from endogenous substrate accounted for about one third of the total, whereas the great part of 12-HETE derived from exogenous arachidonate. These data indicate that under the tested conditions the pool of arachidonate that acts as substrate for cyclo-oxygenase is different from the pool that acts as substrate for lipoxygenase and that the arachidonate released from phospholipids is preferentially utilized by cyclo-oxygenase.     

On the mechanism of the oxygenation of arachidonic acid by human platelet lipoxygenase

Formation of 12L-hydroxy-5,8,10,14-eicosatetraenoic acid from [10L-³H; 3-¹⁴C]arachidonic acid in suspensions of human platelets occurred with extensive loss of tritium and was accompanied by an isotope effect. These experiments showed that there is an antarafacial relation between the elimination of hydrogen from C-10 and insertion of oxygen at C-12 by human platelet lipoxygenase, and that the hydrogen elimination probably occurs as the initial step of the conversion. (Endo) peroxide intermediates formed by the fatty acid cyclo-oxygenase pathway activated platelet lipoxygenase.     

Selective inhibition of prostaglandin endoperoxide thromboxane isomerase by 1-carboxyalkylimidazoles

1-Carboxyalkylimidazoles inhibited the conversion of prostaglandin H₂ to thromboxane B₂ and 12L-hydroxy-5, 8, 10- heptadecatrienoic acid by a partially purified enzyme (prostaglandin endoperoxide thromboxane isomerase) from bovine platelet microsomes. The degree of the inhibition was dependent on the length of carboxyalkyl chain. 1-Carboxyheptylimidazole was the most potent inhibitor, and an almost complete inhibition was obtained at a concentration on the order of 1 μM. The inhibition, as examined with 1-carboxyheptylimidazole, was of noncompetitive type. These 1-carboxyalkylimidazoles did not affect the formation of prostaglandin H₂ from arachidonic acid. Such a selective inhibition was also demonstrated by the reaction of bovine platelet microsomes with arachidonic acid in the presence of 1-carboxyheptylimidazole, resulting in the accumulation of prostaglandin H₂ as an intermediate. Furthermore, a series of 1-alkylimidazoles with no carboxyl group also inhibited the isomerase at higher concentrations. However, the inhibition was not specific for the isomerase; namely, the prostaglandin H₂ formation from arachidonic acid was also affected.     

Increase in 12-lipoxygenase activity in platelets of spontaneously hypertensive rats

12-Lipoxygenase activity in platelets of spontaneously hypertensive rats was investigated. Enzyme activity was measured in the absence and the presence of reduced glutathione. In both assay conditions, 12-lipoxygenase activity in platelets of spontaneously hypertensive rats was significantly higher than that in platelets of normotensive rats. Since 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), a 12-lipoxygenase product of arachidonic acid in platelets, has been reported to be a potent chemoattractant for aortic smooth muscle cells, increase in biosynthesis of 12-HETE in platelets of spontaneously hypertensive rats might contribute to the explanation of pathogenesis of vascular disorder commonly found in hypertension patients.     

High concentrations of arachidonic acid induce platelet aggregation and serotonin release independent of prostagladin endoperoxides and thromboxane A2

We examined platelet aggregation and serotonin release, induced by less than 60 μM arachidonic acid, using washed platelet suspensions in the absense of albumin. The concentration of arachidonic acid use did not cause platelet lysis. Platelet responses induced by less than 20 μM arachidonic acid were inhibited by aspirin, whereas those induced by above 30 μM arachidonic acid were not inhibited, even by both aspirin and 5,8,11,14-eicosatetraynoic acid. Although phosphatidic acid and 1,2-diacylglcerol increased after the addition of arachidonic acid in aspirin-treated platelets, the amounts were not parallel to platelet aggregation. Oleic, linoleic and linolenic acids also induced platelet responses, while palmitic, stearic and arachidic acids did not. EDTA, dibutyryl cyclic AMP, apyrase and creatine phosphate / creatin phosphokinase brought about almost the same effects in platelet responses induced by the unsaturated fatty acids, other than arachodinic acid, as those induced by 40 μM arachodonic acid. These results suggest that the mechanism of the actions of more than 30 μM arachodinic acid on platelets is the same as that of the other unsaturated fatty acids and is independent of prostaglandin endoperoxides, thromboxane A₂ and, perhaps, phosphatidic acid and 1,2-diacylglycerol.     

Synthesis of thromboxane B2 in incubates of dog platelet-rich plasma with arachidonic acid and its inhibition by different drugs

Dog platelets in citrated plasma fail to aggregate upon addition of AA, even though, as demonstrated by bioassay procedures and now by the radioimmunoassay, TxA2 is formed as in case of aggregating human platelets. Imidazole inhibited formation of TxB2 and increased the amounts of PGE2 formed, indicating specific inhibition of thromboxane synthetase. Other drugs tested (benzimidazolamine, compound L8027, indomethacin and isoprenaline) inhibited either cyclo-oxygenase alone, or together with thromboxane synthetase.     

Enzymatic conversion of C21 endoperoxides to thromboxanes and hyroxy acids

ω-Homo and α-homo [³H₈]prostaglandin H₂ were prepared by chemical and enzymatic methods from 5,8,11,14-heneicosatetraynoic acid and 6,9,12,15-heneicosatetraynoic acid. Upon incubation with human platelet microsomes, two products were formed from each endoperoxide, viz. ω-homo thromboxane B₂ plus 12-hydroxy-octadecatrienoic acid and α-homo thromboxane B₂ plus 13-hydroxy-octadecatrienoic acid. In the light of previous results this suggests that the distance between the double bond of the carboxyl side chain and the cyclopentane ring of prostaglandin H₂ may be important for conversion to thromboxanes.     

Aggregation and thromboxane B2 formation in platelets and vascular prostacyclin production from genetically obese rats

Obesity, diabetes, hyperlipidaemia and age are conditions predisposing to atheroscleorosis and arterial occlusion. Recently it has been claimed that increased synthesis of thromboxane A₂ by platelets and decreased synthesis of prostacyclin (PGI₂) by blood vessels play an important role. The “Zucker” rat, a genetically obese animal with hyperlipidaemia, hyperinsulinaemia and normoglycaemia was used to study platelet aggregation, thromboxane (TXB₂) production and aortic PGI₂ synthesis. Two age groups (6–8 months and 14–16 months old) and their homozygote lean controls were used. In the obese rats no increased aggregation was found with ADP, arachidonic acid and collagen. On the contrary platelets from young fatty rats were less sensitive to ADP than platelets from lean young animals. An increase in platelet sensitivity to aggregating agents with age was observed, especially in the obese rats. TXB₂ measured in platelet rich plasma after exposure to ADP, arachidonic acid, arachidonic acid plus ADP and collagen was similar in the fatty and lean animals.Production of PGI₂ from incubated aortic rings was lowest in young lean animals. No differences existed between the other groups of rats studied. Insulin added to aortic rings had no influence on PGI₂ production. It is concluded that age rather than obesity, hyperlipidaemia or hyperinsulinaemia may cause platelet hyperresponsiveness to aggregating agents. Thromboxane and plateletaggregation do not closely correlate. PGI₂ production is not reduced by metabolic alterations, thought to predispose to atherosclerosis.     

Inhibition of human neutrophil arachidonate 5-lipoxygenase by 6, 9-deepoxy-6, 9-(phenylimino)- Δ 6, 8-prostaglandin I1 (U-60257)

6, 9-Deepoxy-6, 9-(phenylimino)-Δ 6, 8-prostaglandin I₁ (U-60257) and its methyl ester (U-56467) are selective inhibitors of leukotriene C and D biosynthesis both $\text{in}$$\text{vitro}$ and $\text{in vivo}$. In this study, we demonstrated that the principal site of inhibition may be arachidonate 5-lipoxygenase, the initial enzyme of leukotriene biosynthesis. U-60257 and its methyl ester block LTB₄ synthesis in human peripheral neutrophils with an ID₅₀ of 1.8 and 0.42 μM respectively. This inhibitory action of U-60257 on neutrophil 5-lipoxygenase can be reduced or reversed by a high concentration of exogenous arachidonic acid. U-60257 at 100 μM has no apparent effect on the following enzymes. 1) cyclooxygenase of sheep vesicular gland or human platelets; 2) 12-lipoxygenase of human platelets and 3) soybean 15-lipoxygenase. Thus, we conclude that U-60257 and its methyl ester potent and selective inhibitors of arachidonate 5-lipoxygenase.     

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.     

Evidence for mediation of acetyl glyceryl ether phosphorylcholine stimulation of adenosine 3′,5′-(cyclic)monophosphate levels in human polymorphonuclear leukocytes by leukotriene B4

Acetyl glyceryl ether phosphorylcholine induces human neutrophil aggregation. Incubation of neutrophils with either prostaglandin I₂, or the cyclic AMP-dependent phosphodiesterase inhibitor, RO 20-1724 before the addition of PAF-acether attenuates subsequent aggregation. Paradoxically, a small elevation in cyclic AMP is observed coincident with the initiation of PAF-acether-stimulated aggregation. The elevation in cyclic AMP in response to PAF-acether is amplified by RO 20-1724, and the magnitude of the response is dependent upon the concentration of PAF-acether. The elevation in cyclic AMP is not due to prostaglandins, because indomethacin actually enhances the elevation in cyclic AMP induced by PAF-acether. The involvement of the neutrophil 5-lipoxygenase, and subsequent leukotriene B₄ synthesis, is suggested by the observation that 5-lipoxygenase inhibitors limit both the elevation in cyclic AMP induced by PAF-acether, and the indomethacin enhancement. This indirect evidence is supported by the fact that leukotriene B₄ itself elevates neutrophil cyclic AMP levels in intact cells, and stimulates the adenylate cyclase in broken cell preparations. Although the elevation in cyclic AMP induced by either PAF-acether or leukotriene B₄ is coincident with the onset of neutrophil aggregation, it is not obligatory for aggregation. The adenylate cyclase inhibitor 2′,5′-dideoxyadenosine blocks the PAF-acether-stimulated increase in cyclic AMP, and actually enhances aggregation. It is suggested that the increase in cyclic AMP observed after the addition of PAF-acether is due to concomitant leukotriene B₄ synthesis, and is not obligatory for neutrophil aggregation, but is actually part of a feed-back regulatory system through which PAF-acether and leukotriene B₄ can limit their own activity in neutrophils.     

Involvement of prostaglandin E2 synthesis in the intestinal secretory action of Escherichia coli heat-stable enterotoxin II

Abstract The prostaglandin response of mouse intestinal epithelial cells after exposure to Escherichia coli heat-stable enterotoxin II was examined. The quantity of prostaglandin E₂ produced by the intestinal cells was directly related to the dose of heat-stable enterotoxin II. The change in the amount of prostaglandin E₂ over time correlated to that of the volume of fluid released into the intestinal lumen. We then demonstrated that administration of heat-stable enterotoxin II into the intestinal loops of mice induced elevation of arachidonic acid and phosphatidic acid levels in intestinal epithelial cells. These results show that heat-stable enterotoxin II stimulates arachidonic acid metabolism in intestinal epithelial cells and that the synthesized prostaglandin E₂ functions as a mediator of fluid secretion induced by this enterotoxin.     

Participation of Prostaglandin E2 in Dopamine D2 Receptor-Dependent Potentiation of Arachidonic Acid Release

Stimulation of dopamine D2 receptors potentiates Ca2+ ionophore- or ATP-induced arachidonic acid (AA) release in D2 receptor cDNA-transfected Chinese hamster ovary (CHO) cells [CHO(D2)]. By using a combination of chromatographic, biochemical, and radioimmunochemical techniques, we show here that prostaglandin (PG) E2 is a major product of AA metabolism in CHO(D2) cells stimulated with the Ca2+ ionophore A23187. Formation of this PG was markedly increased by the concomitant application of quinpirole, a D2 receptor agonist. In addition, PGE2 enhanced D2-dependent amplification of AA release, either when it was added (EC50 = 100 nM) or when it was produced endogenously, as shown by experiments carried out with the cyclooxygenase inhibitor indomethacin. The results suggest that PGE2 may participate in D2 receptor-mediated potentiation of AA release in CHO(D2) cells. They also support a functional role for this PG in the modulation of dopaminergic transmission in areas of the CNS, such as amygdala and hypothalamus, where high levels of both PGE2 and dopamine D2 receptors are found.     

Biosynthesis of thromboxane B2: Assay, isolation, and properties of the enzyme system in human platelets

The microsomal fraction of human platelets catalyzed the conversion of arachidonic acid to an unstable platelet-aggregating factor and a hydrolyzed product on thin-layer chromatography (TLC). This product was isolated on TLC, purified by silica gel column chromatography and identified by combined gas chromatography-mass spectrometry as the hemiacetal derivative of 8-(1-hydroxy-3-oxopropyl)-9, 12L-dihydroxy-5, 10-heptadecatrienoic acid (thromboxane B₂). The enzymatic activity was dependent upon methemoglobin and tryptophan as cofactors. Reduced glutathione had no effect either alone or in combination with other cofactors. Methemoglobin could be replaced by hematin or hemin; and tryptophan by 3-indolacetic acid or catecholamines. The apparent requirement for methemoglobin is due to the reductive activity of ferriprotoporphyrin IX. The reaction, however, catalyzed by the ferriprotoporphyrin IX in the thromboxane synthesizing system is different from that described for the decomposition of lipid peroxides. Certain transition metals and hydrogen donors, such as hydroquinone and ascorbate, which have been shown to stimulate the catalytic activity of ferriprotoporphyrin IX in the decomposition of 15-hydroperoxy-prostaglandin E₁ are inhibitors of thromboxane B₂ formation. This enzyme preparation also transformed eicosa-8, 11, 14-trienoic acid to an unknown product on TLC. The enzyme system was rapidly inactivated upon incubation in the reaction mixture.     

Hydroperoxy fatty acid formation in selenium deficient rat platelets: coupling of glutathione peroxidase to the lipoxygenase pathway

After incubation with [1-14C]-arachidonic acid, washed platelets from selenium deficient rats produced a sevenfold greater amount of 12-hydroperoxytetraenoic acid than platelets from control animals. When stimulated with either arachidonic acid or t-butyl-hydroperoxide, antimycin-A1 treated platelets from the deficient rats also converted markedly lower amounts of [1-14C]-glucose to [14C]-CO2 than platelets from control rats. These results indicate a significant role for platelet selenium-dependent glutathione peroxidase in the enzymatic reduction of platelet-produced hydroperoxides.     

Studies on the urinary excretion of thromboxane B2 in Zellweger patients and control subjects: evidence for a major role for peroxisomes in the β-oxidative chain-shortening of thromboxane B2

In this paper we studied the urinary excretion of thromboxane B₂ and its β-oxidation product 2,3-dinor-thromboxane B₂ in urines from control subjects and four Zellweger patients, which lack morphologically distinguishable peroxisomes. In the urine of three classical Zellweger patients we found a ratio of 2,3-dinor-thromboxane B₂/thromboxane B₂ of 0.35, 0.48 and 0.62 respectively, whereas in healthy children and adults values were found of 3.1–10 and 5.5–40 respectively. These data strongly suggest that peroxisomes are a major site for β-oxidation of thromboxane B₂.     

Inhibitory effect of GBH on platelet aggregation through inhibition of intracellular Ca2+ mobilization in activated human platelets

Geiji-Bokryung-Hwan (GBH) was studied on antiplatelet activity in human platelet suspensions. GBH consists of the 5 herbs Cinnamomi Ramulus, Poria Cocos, Mountan Cortex Radicis, Paeoniae Radix, and Persicae Semen, which have been used in herbal medicine for thousands of years for atherosclerosis. The mechanism involved in the antiplatelet activity of GBH in human platelet suspensions was investigated. GBH inhibited platelet aggregation and Ca2+ mobilization in a concentration-dependent manner without increasing intracellular cyclic AMP and cyclic GMP. GBH had no inhibitory effect on thromboxane B2 (TXB2) production in cell-free systems. Collagen-related peptide (CRP)-induced Ca2+ mobilization is regulated by phospholipase C-2 (PLC-gamma2) activation. We evaluated the effect of GBH on tyrosine phosphorylation of PLC-gamma2 and the production of inositol-1,4,5-trisphosphate (IP3). GBH at concentrations that inhibited platelet aggregation and Ca2+ mobilization had no effects on tyrosine phosphorylation of PLC-gamma2 or on the formation of IP3 induced by CRP. Similar results were obtained with thrombin-induced platelet activation. GBH inhibited platelet aggregation and Ca2+ mobilization induced by thrombin without affecting the production of IP3. We then evaluated the effect of GBH on the binding of IP3 to its receptor. GBH at high concentrations partially blocked the binding of IP3 to its receptor. Therefore, the results suggested that GBH suppresses Ca2+ mobilization at a step distal to IP3 formation. GBH may provide a good tool for investigating Ca2+ mobilization.