An improved malondialdehyde assay for estimation of thromboxane synthase activity in washed human blood platelets

After stimulation of the washed human blood platelets by arachidonic acid (AA), the concurrent evaluations for formed malondialdehyde (MDA) measured by the common photometrical thiobarbituric acid (TBA) method, and for thromboxane B2 (TXB2) measured by gas chromatography, revealed that the formed MDA exceeded the amount of TXB2 on a molar base. However, MDA and TXB2 originating from thromboxane synthase activity should be produced in approximately equimolar amounts. By treatment of the stimulated platelet samples with stannous chloride it is possible to reduce all peroxidized products of AA which generate MDA otherwise during the TBA reaction and to estimate MDA and TXB2 in a ratio of nearly 1:1. The stannous chloride treatment does not destroy the MDA and does not influence the TBA reaction with MDA. Therefore the simple and quick TBA method can be used after stannous chloride treatment for estimation of thromboxane synthase activity in AA stimulated washed human platelets.     

Increased production of ovarian thromboxane in gonadotropin-treated immature rats: Relationship to the ovulatory process

Thromboxane (TX) B₂, a stable metabolic product of hydrolysis of TXA₂, was measured by radioimmunoassay in tissue extracts of ovaries of immature rats pretreated with pregnant mare's serum gonadotropin and human chorionic gonadotropin. Ovarian concentrations of TXB₂ increased before, and remained elevated after, the time of ovulation. In a subsequent study, ovulation was inhibited in a dose-dependent fashion by a reported TXA₂ receptor antagonist, AH23848. Nevertheless, inhibition of the preovulatory rise in synthesis of TXB₂ by furegrelate (a thromboxane synthetase inhibitor) did not prevent ovulation. Nor was the blockade of ovulation caused by indomethacin (a cyclooxygenase inhibitor) reversed by a TXA₂ mimetic (U-46619). It does not appear that a preovulatory increase in ovarian thromboxane is an obligatory component of the ovulatory mechanism of gonadotropin-primed immature rats.     

Kinetic and structural evidence for the identification of 11-hydroxythromboxane B2 dehydrogenase as cytosolic aldehyde dehydrogenase

We have recently purified 11-hydroxythromboxane B₂ dehydrogenase from porcine kidney and identified it as cytosolic aldehyde dehydrogenase (EC 1.2.1.3) based on amino acid analysis and other protein characteristics. In the present paper we have studied the catalytic interaction of thromboxane B₂ (TXB₂) with different aldehyde substrates and a potent aldehyde dehydrogenase inhibitor, disulfiram. TXB₂ was a competitive inhibitor of the aldehyde dehydrogenase reaction in assays with 3,4-dihydroxyphenylacetaldehyde, a high affinity substrate. The conversion of TXB₂ to 11-dehydro-TXB₂ was also inhibited by propanal and disulfiram.

The protein characteristics of the enzyme have also been further studied. The native enzyme is a tetramer and has an isoelectric point of 7.0 which is comparable with that of cytosolic aldehyde dehydrogenases from other species. Taken together the present data further indicate that 11-hydroxythromboxane B₂ dehydrogenase is identical with cytosolic aldehyde dehydrogenase and that substrates and inhibitors of aldehyde dehydrogenase interact with thromboxane metabolism in vitro.     

Prostanoid formation in primary astroglial cell cultures: Ca-dependency and stimulation by A 23187, melittin and phospholipases A2 and C

Prostaglandin (PG) and thromboxane B₂ (TXB₂) biosynthesis was studied in cultured astrocytes from neonatal rat brain hemispheres. After two weeks of cultivation, prostanoids were formed with the spectrum: PGD₂ > TXB₂ > PGF₂ > PGE₂, as measured by specific radioimmunoassays. Under basal conditions PGD₂ biosynthesis (9.55 ng/mg protein/15 min) was in the same order of magnitude as the sum of the other prostanoids. The formation of prostanoids was stimulated in a concentration dependent manner (up to 6–10 fold) by the calcium ionophore A 23187 (0.01–10 μM) as well as by melittin (0.01–5 μg/ml), phospholipase A₂ (10–40 U/ml) and phospholipase C (0.01–1 U/ml). Basal and evoked PG and TXB₂ biosynthesis depended on the availability of Ca²⁺, as demonstrated in Ca²⁺ free incubation medium containing Na₂EDTA (1 μM), or with verapamil (100 μM) and 3,4,5-trimethoxybenzoic acid-8-(diethylamino)-octylester-HCl (TMB-8, 1–100 μM). Indomethacin (10 μM), mepacrine (100 μM) and p-bromophenacylbromide (50 μ M) inhibited basal and evoked PG formation. Thin-layer chromatography (TLC) detection after incubation of the cells with [³H]arachidonic acid (1 μCi/ml, for 60 min) confirmed the results obtained by radioimmunoassay. Incubation of [³H]arachidonic acid labelled cells with inonophore or phospholipases, followed by lipid extraction and TLC, showed that A 23187 liberated [³H]arachidonic acid predominantly from phosphatidylethanolamine, whereas phospholipase A₂ and C reduced mainly the labelling of the phosphatidyl-inositol/-choline fraction. Potassium depolarization of the cells did not enhance prostanoid formation. Similarly, drugs with affinity to - or β-adrenoceptors, or to dopamine-, 5-hydroxytryptamine-, muscarine-, histamine-, glutamate-, aspartate-, GABA, adenosine- and opioid-receptors failed to stimulate prostanoid biosynthesis. Also compounds like angiotensin, bradykinin and thrombin were ineffective in this respect.

In conclusion, our results confirm that cultured astrocytes possess the complete pattern of enzymes necessary for prostanoid formation and hence might play a crucial role in brain prostanoid biosynthesis. Stimulation of prostanoid biosynthesis involves Ca²⁺-dependent activation of phospholipase A₂, cyclooxygenase reaction and further PG metabolism. However, the endogenous stimulus for enhanced prostanoid synthesis in the brain still has to be established.     

Inhibition of the human platelet cyclooxygenase response by the naturally occurring phenazine derivative, 1-hydroxyphenazine

The phenazine derivative, 1-hydroxyphenazine (OHP), is produced in vivo by Pseudomonas aeruginosa, an organism that colonises the airways of patients with cystic fibrosis. While known to inhibit leukotriene production by human neutrophils, the effects of OHP on cyclooxygenase pathways have not previously been reported. We used [³H]arachidonic acid (AA) under conditions of concurrent labelling-stimulation or pre-labelling for one hour followed by stimulation to determine the effects of OHP on the production of cyclooxygenase metabolites by human platelets stimulated with the calcium ionophore, A23187. Thromboxane B₂ (TxB₂) and 12-hydroxyheptadecatrienoic acid (HHT) production was inhibited in a dose-dependent manner by OHP using either pre-labelled or concurrently labelled platelets. However, production of 12-hydroxyeicosatetraenoic acid (12-HETE) was not diminished. Determination of the amount of total free label (AA + non-esterified AA metabolites) after stimulation of pre-labelled platelets indicated a dose-dependent inhibition of the release of AA from phospholipid by OHP. This was reflected in a corresponding increase in phospholipid AA content. These data indicate that phenazine derivatives of bacterial origin exhibit complex interactions with pathways of arachidonic acid metabolism in host cells. These effects may prove to be of pharmacological importance.     

Arachidonic acid metabolism in thrombocytes and vascular tissues of turkeys

Turkeys are hypertensive compared to mammals of similar size. In vitro synthesis of thrombocyte thromboxane B₂ (TxB₂), 12L-hydroxy-5, 8, 10 heptadecatrienoic acid (HHT), 12L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) and aortic prostaglandin (PG) production was studied in one to ten month old domestic white turkeys. Compared to normal human platelets, TxB₂ production was increased (55.4 vs. 31.4%) and HETE production was markedly reduced (6.5 vs. 34.6%) in control thrombocytes. Similar to human platelets in which cyclooxygenase inhibition with aspirin results in an increase in HETE production, block of the thrombocyte enzyme with aspirin doubled the production of HETE. In vitro conversion of radiolabeled arachidonic acid (AA) showed that the primary PG produced by turkey aorta was PGE₂. A 6-keto immunoreactive PG was present which comigrated with authentic 6-keto PGF₁, but failure of the aortic supernatant to inhibit adenosine diphosphate or AA induced platelet aggregation suggested that PGI₂ was not produced. The vasodepressor potency of PGE₁, PGE₂ and PGI₂ was altered in awake turkeys with PGE₁ and PGE₂ having five times the hypotensive effect as PGI₂. In addition, conversion of AA to PGE₂ by aorta in one month turkeys was greater (17.3 vs. 9.2%) than in ten month old turkeys. Systemic arterial pressure was increased in the ten month old turkeys (188 mmHg) compared to one month old turkeys (143 mmHg). Thus, both vascular AA metabolism and the vasodepressor potencies of PGE₂ and PGI₂ are altered and the activity of the lipoxygenase pathway in thrombocytes is limited in the turkey.     

The Action of Hydrogen Peroxide on the Formation of Thiobarbituric Acid-Reactive Material From Microsomes, Liposomes Or From Dna Damaged By Bleomycin Or Phenanthroline. Artefacts in the Thiobarbituric Acid Test

Incubation of rat-liver microsomes, previously azide-treated to inhibit catalase, with H₂O₂ caused a loss of cytochrome P-450 but not of cytochrome b₅. This loss of P-450 was not prevented by scavengers of hydroxyl radical, chain-breaking antioxidants or metal ion-chelating agents. Application of the thiobarbituric acid (TBA) assay to the reaction mixture suggested that H₂O₂ induces lipid peroxidation, but this was found to be due largely or completely to an effect of H²O₂ on the TBA assay. By contrast, addition of ascorbic acid and Fe(III) to the microsomes led to lipid peroxidation and P-450 degradation: both processes were inhibited by chelating agents and chain-breaking antioxidants, but not by hydroxyl radical scavengers. H₂O₂ inhibited ascorbate/Fe (III)-induced microsomal lipid peroxidation, but part of this effect was due to an action of H₂O₂ in the TBA test itself. H₂O₂ also decreased the colour measured after carrying out the TBA test upon authentic malondialdehyde, tetraethoxypropane, a DNA-Cu²⁺/o-phenanthroline system in the presence of a reducing agent, ox-brain phospholipid liposomes in the presence of Fe(III) and ascorbate, or a bleomycin-iron ion/DNA/ascorbate system. Caution must be used in interpreting the results of TBA tests upon systems containing H₂O₂.     

孕康口服液对肾虚-黄体抑制型先兆流产大鼠的安胎作用

目的:孕康口服液为已上市中成药,为进一步评价其药效,本实验通过建立肾虚-黄体抑制型先兆流产模型,观察孕康口服液的安胎作用。方法:60只妊娠大鼠随机分为正常对照组(NC),模型组(MG),地屈孕酮组(DT,3.02 mg/kg),孕康口服液低剂量组(YK-L,4 ml/kg)、中剂量组(YK-M,6 ml/kg)、高剂量组(YK-H,9 ml/kg),每组10只。自妊娠第1日,每日上午各给药组按规定剂量灌予受试药,NC组、MG组给予等体积的纯化水,连续10 d;每天下午灌胃造模,除NC组给予纯化水外,其余各组按450 mg/kg体质量灌胃羟基脲,连续9 d,第10日按4.0 mg/kg体质量灌胃米非司酮。妊娠第9日,测定各组大鼠背温、抓力、痛阈、自主活动等行为体征;妊娠第11日,各组腹主动脉取血,测定血清雌二醇(E₂)、孕酮(P)、血栓素B₂(TXB₂)水平;摘取卵巢、连胎子宫,观察胚胎个数和直径,计算卵巢、连胎子宫指数。结果:与NC组比较,MG组背温、抓力、痛阈、自主活动次数、胚胎个数、胚胎直径、连胎子宫指数和血清E₂、P、TXB₂水平均显著降低(P＜0.05,0.01)。与MG组比较,孕康口服液各剂量组背温、抓力、胚胎个数、胚胎直径和血清E₂、P水平均显著升高(P＜0.05,0.01);YK-M、YK-H组痛阈、自主活动、连胎子宫指数显著升高(P＜0.05);YK-H组血清TXB₂水平明显升高(P＜0.05)。结论:孕康口服液对肾虚-黄体抑制导致的先兆流产大鼠具有明确的补肾安胎作用,其机制可能与升高血清E₂、P、TXB₂水平,改善肾虚体征和提高胚胎质量有关。     

Bleomycin-Iron Damage To Dna With Formation Of 8-Hydroxydeoxyguanosine and Base Propenals. Indications That Xanthine Oxidase Generates Superoxide From Dna Degradation Products

Bleomycin, in the presence of ferric salts, oxygen and a suitable reductant, degrades DNA with the release of base propenals, detected as thiobarbituric acid (TBA) reactivity, and the formation of 8-hydroxydeo-xyguanosine (80HdG) detected by HPLC. When xanthine oxidase is added to the incubated mixture of DNA degradation products, TBA-reactivity is destroyed but 80HdG formation is increased. EPR Spin trapping experiments show that hydroxyl radicals (OH) are formed in the reaction mixture and can be inhibited by the inclusion of either superoxide dismutase or catalase. These findings suggest that the base propenals and possibly malondialdehyde, formed from them, are aldehydic substrates for xanthine oxidase and, the product of this reaction is superoxide (O₂^-) and hydrogen peroxide (H₂O₂). Thus, TBA reactivity is destroyed in the formation of O₂^- and H₂O₂ which stimulate further oxidative damage to DNA resulting in increased 8OHdG formation.     

Acetyl glyceryl ether phosphorylcholine (platelet-activating factor) mediates heightened metabolic activity in macrophages: Studies on PGE, TXB2 and O2 production, spreading, and the influence of calmodulin-inhibitor W-7

The phospholipid mediator AGEPC (acetyl glyceryl ether phosphorylcholine) was examined for its effects on guinea pig peritoneal macrophages. At a concentration of 10⁻⁹ -10⁻⁶ M, AGEPC evoked release of prostaglandin E (PGE) and thromboxane B₂ (TXB₂) from albumin-elicited macrophages. It also triggerd generation of O⁻₂ by Corynebacterium parvum-induced cells. Moreover, it caused augmented spreading of macrophages. The calmodulin antagonis W-7 attenuated AGEPC-mediated O⁻₂ production and cell spreading whereas prostanoid synthesis was enhanced. These novel actions of AGEPC on the major cellular component of the inflammatory process attest to its role as a potent mediator of immunoinflammatory responses.     

Blood eicosanoids and immune indices during fasciolosis in water buffaloes

The effects of trickle infections of water buffaloes with Fasciola hepatica (60 metacercariae daily during a period of 20 days) on the blood plasma levels of prostaglandin E₂ (PGE₂), 6-keto-prostaglandin F₁ (6-keto-PG F₁) and thromboxane B₂ (TXB₂) were assessed. F. hepatica specific IgG and T- and B-lymphocyte ratios were evaluated as indicators of the immune response. Although the applied mode of infection did not result in clinical disease, changes in the plasma eicosanoid pattern were observed. Plasma PGE₂ values were significantly elevated in the infected water buffaloes 11 weeks post-infection (w.p.i.). In contrast, transiently but significantly lower TXB₂ values than in the uninfected controls were recorded in the phase of chronic fasciolosis. Plasma 6-keto-PGF₁ values were not considerably altered by the infection throughout the study period. F. hepatica-specific IgG were detected from 4 to 21 w.p.i. The proportion of peripheral T- and B-lymphocytes shifted towards B-cells from 2 to 12 w.p.i., gradually returning to control values afterwards. Although the water buffaloes appeared to be rather resistant to trickle infection with F. hepatica, moderate changes in plasma eicosanoid patterns were observed, indicating tissue damage and/or inflammation. Induction of the immune response could be monitored by an increase of F. hepatica-specific IgG, which was paralleled by a relative increase of the B-lymphocyte population.     

Stimulation of eicosapentaenoic acid metabolism in washed human platelets by 12-hydroperoxyeicosatetraenoic acid

Washed human platelets were not able to convert eicosapentaenoic acid (EPA) to thromboxane B3 (TXB3) and 12-hydroxyeicosapentaenoic acid (AA) to washed human platelets induced conversion of EPA to TXB3 and 12-HEPE. Esculetin, a specific inhibitor of 12-lipoxygenase, prevented the effect of AA, but cyclooxygenase inhibitor did not. The conversion of AA to TXB2 was not affected by the same dose of esculetin. These data suggest that products of AA formed by 12-lipoxygenase in human platelets have stimulatory effects on EPA metabolism. When AA was preincubated with washed human platelets, its effect on EPA conversion was reduced, suggesting that a labile product of AA formed by 12-lipoxygenase is involved in the facilitation of EPA metabolism. Addition of 12-hydroperoxyeicosatetraenoic acid directly to washed human platelets caused dose-dependent synthesis of TXB3 and 12-HEPE, while addition of 12-hydroxyeicosatetraenoic acid had no effect. Thus, 12-hydroperoxyeicosatetraenoic acid formed from AA promotes the metabolism of EPA in washed human platelets.     

Role of contaminating platelets in thromboxane synthesis in primary cultures of human umbilical vein endothelial cells

Previous studies suggested that cultured human endothelial cells metabolize arachidonic acid to thromboxane A2. When primary cultures of human umbilical vein endothelial cells were incubated with 14C-arachidonic acid and the 14C-metabolites resolved by reverse phase high pressure liquid chromatography, radioactive products were observed that comigrated with 6-keto-prostaglandin F1alpha and thromboxane B2, the degradation products of prostacyclin and thromboxane A2, respectively. Since platelets synthesize thromboxane A2, the present study examined the hypothesis that adherent platelets may contaminate the primary cultures of human umbilical vein endothelial cells and be responsible for thromboxane B2 production. Confluent primary cultures or passaged cells were stimulated with histamine (10(-5) M). Incubation buffer was analyzed by specific radioimmunoassays for 6-keto-prostaglandin F1alpha and thromboxane B2. The production of thromboxane B2 decreased in the passaged cells (207 +/- 44 pg/ml versus 65 +/- 12 pg/ml; primary versus passaged cells). A moderate decrease in the yield of 6-keto-prostaglandin F1alpha was measured in the passaged cells compared to the primary cultures (3159 +/- 356 pg/ml versus 1678 +/- 224 pg/ml, primary versus passaged cells). If the primary cultures were incubated with human platelet-rich plasma for 30 min prior to stimulation with histamine, the amount of thromboxane B2 increased approximately 10-fold. In an additional experiment, sub-confluent primary cells were incubated with platelet-rich plasma for 30 min, washed to remove non-adherent platelets, and allowed to reach confluency. Confluent cells were then passaged and stimulated with histamine. The amount of thromboxane B2 was not significantly different from that obtained with passaged cells that had not been incubated with platelet-rich plasma during the primary culture (83 +/- 15 pg/ml versus 65 +/- 12 pg/ml, respectively). If the cyclooxygenase inhibitor indomethacin was included in the incubations, the amounts of both thromboxane B2 and 6-keto-prostaglandin F1alpha decreased. In contrast, the thromboxane A2 synthase inhibitor dazoxiben blocked thromboxane production and had no effect on the amount of 6-keto-prostaglandin F1alpha. Light microscopy revealed the presence of adherent platelets in primary cultures with and without platelet-rich plasma but no platelets were observed in any group of passaged cells. Histofluorescence for platelet serotonin indicated the presence of platelets only in primary cultures of human umbilical vein endothelial cells or in cultures pre-incubated with platelet-rich plasma. These studies suggest that primary cultures of human umbilical vein endothelial cells contain adherent platelets that contribute to thromboxane synthesis.     

Oxidant-mediated AA release from astrocytes involves cPLA(2) and iPLA(2)

Excessive generation of reactive oxygen species (ROS) in the central nervous system (CNS) is a leading cause of neuronal injury. Despite yet unknown mechanisms, oxidant compounds such as H₂O₂ have been shown to stimulate the release of arachidonic acid (AA) in a number of cell systems. In this study, H₂O₂ and menadione, a compound known to release H₂O₂ intracellularly, were used to examine the phospholipases A₂ (PLA₂) responsible for AA release from primary murine astrocytes. Both H₂O₂ and menadione dose-dependently stimulated AA release, and the release mediated by H₂O₂ was completely inhibited by catalase. H₂O₂ also stimulated phosphorylation of extracellular signal-regulated kinases (ERK1/2) and cytosolic phospholipase A₂ (cPLA₂). However, complete inhibition of cPLA₂ phosphorylation by U0126, an inhibitor for mitogen-activated protein kinase kinase (MEK) and GF109203x, a nonselective PKC inhibitor preferring the conventional and novel isoforms, only reduced H₂O₂-stimulated AA release by 50%. MAFP, a selective, active, site-directed, irreversible inhibitor of both cPLA₂ and the Ca²⁺-independent iPLA₂, nearly completely inhibited H₂O₂-mediated AA release; but, HELSS, a potent irreversible inhibitor of iPLA₂, only inhibited H₂O₂-mediated AA release by 40%. Along with the observation that H₂O₂-mediated AA release was only partially inhibited upon chelating intracellular Ca²⁺ by BAPTA, these results indicate the involvement of both cPLA₂ and iPLA₂ in H₂O₂-mediated AA release in murine astrocytes.     

Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury

Increasing appreciation of the causative role of oxidative injury in many disease states places great importance on the reliable assessment of lipid peroxidation. Malondialdehyde (MDA) is one of several low-molecular-weight end products formed via the decomposition of certain primary and secondary lipid peroxidation products. At low pH and elavated temperature, MDA readily participates in nucleophilic addition reaction with 2-thiobarbituric acid (TBA), generating a red, fluorescent 1:2 MDA:TBA adduct. These facts, along with the availability of facile and sensitive methods to quantify MDA (as the free aldehyde or its TBA derivative), have led to the routine use of MDA determination and, particularly, the “TBA test” to detect and quantify lipid peroxidation in a wide array of sample types. However, MDA itself participates in reactions with molecules other than TBA and is a catabolic substrate. Only certain lipid peroxidation products generate MDA (invariably with low yields), and MDA is neither the sole end product of fatty peroxide formation and decomposition nor a substance generated exclusively through lipid peroxidation. Many factors (e.g., stimulus for and conditions of peroxidation) modulate MDA formation from lipid. Additional factors (e.g., TBA-test reagents and constituents) have profound effects on test response to fatty peroxide-derived MDA. The TBA test is intrinsically nonspecific for MDA: nonlipid-related materials as well as fatty peroxide-derived decomposition products other than MDA are TBA positive. These and other considerations from the extensive literature on MDA, TBA reactivity, and oxidative lipid degradation support the conclusion that MDA determination and the TBA test can offer, at best, a narrow and somewhat empirical window on the complex process of lipid peroxidation. The MDA content and/or TBA reactivity of a system provides no information on the precise structures of the “MDA precursor(s),” their molecular origins, or the amount of each formed. Consequently, neither MDA determination nor TBA-test response can generally be regarded as a diagnostic index of the occurrence/extent of lipid peroxidation, fatty hydroperoxide formation, or oxidative injury to tissue lipid without independent chemical evidence of the analyte being measured and its source. In some cases, MDA/TBA reactivity is an indicator of lipid peroxidation; in other situations, no qualitative or quantitative relationship exists among sample MDA content, TBA reactivity, and fatty peroxide tone. Utilization of MDA analysis and/or the TBA test and interpretation of sample MDA content and TBA test response in studies of lipid peroxidation require caution, discretion, and (especially in biological systems) correlative data from other indices of fatty peroxide formation and decomposition.     

Synergistic effect of cadmium chloride and acetaldehyde on cytotoxicity and its prevention by quercetin and glycyrrhizin

Cadmium chloride at concentrations of 10-50mM and acetaldehyde (AA) at 1-5mM showed synergistic toxic effects on V79 cells in vitro. Furthermore, synergistic effects of these chemicals were also observed in mutagenicities of the Hprt gene within certain dose ranges (cadmium chloride 5-10mM, and AA 1-2.5mM). Moreover, lipid peroxide formation, malondialdehyde (MDA) formation, detected by 2-thiobarbituric acid (TBA) reaction and the mitochondrial membrane potentials detected by rhodamine 123 uptake were significantly increased with the combined effect of cadmium and AA in V79. Thus, the cytotoxicity and genotoxicity displayed by combination of these chemicals can be considered to be associated with oxidative stress. Further, these effects were efficiently reduced by quercetin and less efficiently with glycyrrhizin.     

Differential effects of prostaglandins and arachidonic acid on gastric circulation and oxygen consumption

Experiments were carried out on anesthetized dogs to compare the effects of prostaglandin E₂ (PGE₂), prostacyclin (PGI₂) and arachidonic acid (AA) administered intraarterially on gastric blood flow and oxygen consumption during constant arterial pressure perfusion and constant flow perfusion of the stomach. Both PGE₂ and PGI₂ increased total blood flow and oxygen consumption both in the resting stomach and following histamine stimulation although the effects of PGE₂ on the oxygen consumption in stimulated stomach were not statistically significant. On the contrary, AA decreased both gastric blood flow and oxygen consumption in the histamine stimulated stomach. To determine if these compounds can influence gastric oxygen consumption independently of their effects on blood flow, the experiments with constant flow perfusion were performed. Both PGE₂ and PGI₂ decreased both the perfusion pressure and oxygen consumption in the resting as well as in the histamine-stimulated stomach whereas AA increased perfusion pressure and decreased oxygen consumption during histamine administration. Effects of AA were blocked by indomethacin suggesting that not AA itself but some of its metabolites, most likely thromboxanes were responsible for the hemodynamic and metabolic changes resulting from the contraction of gastric arterioles and precapillary sphincters. On the contrary, both PGE₂ and PGI₂ caused gastric hyperemia and an increase in oxygen consumption in the resting stomach, but decreased the latter parameter in the stimulated stomach, most probably as a result of secretory inhibition overcoming direct vascular effects of these compounds.     

Binding of prostacyclin by plasma glycoproteins

The binding of prostacyclin (PGI₂) to plasma proteins and the resulting increase in PGI₂ stability was investigated. Using gel filtration to separate bound and free PGI₂, we have found that Cohn Fraction VI can bind PGI₂, and retard its hydrolysis to 6-keto-PGF₁ (6KPGF₁). The biological activity of the bound PGI₂ correlated well with the quantity of bound PGI₂, measured as 6KPGF₁ by RIA. Fraction VI bound a greater percentage of PGI₂ than the other eicosanoids tested (i.e., PGI₂ > TXB₂ > LTB₄ > PGE₁ > PGF₂). The PGI₂ binding activity of Fraction VI was lost after neuraminidase treatment. Our data suggest that Fraction VI glycoproteins may play an important role in the binding and stabilization of PGI₂ by plasma proteins.     

Prostanoid production in Saccharomyces cerevisiae provides a novel assay for nonsteroidal anti-inflammatory drugs

Prostanoids are a large family of lipid mediators originating from prostaglandin H synthase (PGHS) activity on the 20-carbon polyunsaturated fatty acids dihomo-γ-linolenic acid (DGLA), arachidonic acid (AA) and eicosapentaenoic acid. The two mouse PGHS isoforms, PGHS-1 and PGHS-2, were expressed in Saccharomyces cerevisiae (yeast), as was a signal-peptide-deleted version of PGHS-1 (PGHS-1MA). PGHS-1 showed high activity with both AA and DGLA as substrate, whereas PGHS-2 activity was high with DGLA but low with AA. Signal peptide removal reduced the activity of PGHS-1MA by >50% relative to PGHS-1, but the residual activity indicated that correct targeting to the lumen of the endoplasmic reticulum may not be necessary for enzyme function. Coexpression of PGHS-1 with cDNAs encoding mouse prostaglandin I synthase and thromboxane A synthase, and with Trypanosoma brucei genomic DNA encoding prostaglandin F synthase in AA-supplemented yeast cultures resulted in production of the corresponding prostanoids, prostaglandin I₂, thromboxane A₂ and prostaglandin F_2α. The inhibitory effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on prostanoid production were tested on yeast cells expressing PGHS-1 in AA-supplemented culture. Dose-dependent inhibition of prostaglandin H₂ production by aspirin, ibuprofen and indomethacin demonstrated the potential utility of this simple expression system in screening for novel NSAIDs.     

Glutathione disulfide production during arachidonic acid oxygenation in human platelets

Washed human platelets stimulated with 50 microM sodium arachidonate rapidly accumulated glutathione disulfide to a peak concentration of 0.620 nmole per 10(9) cells, 200% of control (unstimulated) levels. Total glutathione remained unchanged. The rise in glutathione disulfide was transitory, returning to control values within 30 seconds in aggregating platelets. Similar findings were observed in washed platelets aggregated with 5 U/ml thrombin. Platelet aggregation was not necessary for the generation of glutathione disulfide. However, cyclooxygenase activity was necessary for the generation of glutathione disulfide. Aspirin treated platelets aggregated with thrombin demonstrated no thromboxane B2 production and no glutathione disulfide generation. Dose response studies with both agonists demonstrated a direct relationship between the amount of thromboxane B2 produced and the amount of glutathione disulfide generated by stimulated platelets. During the conversion of arachidonic acid to thromboxane B2, unesterified arachidonic acid is oxygenated to prostaglandin G2 which is subsequently reduced to prostaglandin H2. Both reactions are catalyzed by the enzyme prostaglandin H synthase. Our data support the hypothesis that glutathione is an important supplier of reducing equivalents to prostaglandin H synthase during the production of prostaglandin H2 in human platelets.