Thromboxane B2 Generation Research Articles

Cromoglycate drugs suppress eicosanoid generation in U937 cells by promoting the release of Anx-A1

Using biochemical, epifluorescence and electron microscopic techniques in a U937 model system, we investigated the effect of anti-allergic drugs di-sodium cromoglycate and sodium nedocromil on the trafficking and release of the anti-inflammatory protein Annexin-A1 (Anx-A1) when this was triggered by glucocorticoid (GC) treatment. GCs alone produced a rapid (within 5min) concentration-dependent activation of PKCα/β (Protein Kinase C; EC 2.7.11.13) and phosphorylation of Anx-A1 on Ser27. Both phosphoproteins accumulated at the plasma membrane and Anx-A1 was subsequently externalised thereby inhibiting thromboxane (Tx) B2 generation. When administered alone, cromoglycate or nedocromil had little effect on this pathway however, in the presence of a fixed sub-maximal concentration of GCs, increasing amounts of the cromoglycate-like drugs caused a striking concentration-dependent enhancement of Anx-A1 and PKCα/β phosphorylation, membrane recruitment and Anx-A1 release from cells resulting in greatly enhanced inhibition of TxB2 generation. GCs also stimulated phosphatase accumulation at the plasma membrane of U937 cells. Both cromoglycate and nedocromil inhibited this enzymatic activity as well as that of a highly purified PP2A phosphatase preparation. We conclude that stimulation by the cromoglycate-like drugs of intracellular Anx-A1 trafficking and release (hence inhibition of eicosanoid release) is secondary to inhibition of a phosphatase PP2A (phosphoprotein phosphatase; EC 3.1.3.16), which probably forms part of a control loop to limit Anx-A1 release. These experiments provide a basis for a novel mechanism of action for the cromolyns, a group of drugs that have long puzzled investigators.

Serum thromboxane B2 (TXB2) Determination is Influenced by Sample Incubation Temperature in Healthy Beagle Dogs

The measurement of serum thromboxane B2 (TXB2) production by platelets is a specific test for assessment of platelet cyclooxygenase (COX-1) activity following administration of non-steroidal anti-inflammatory drugs (NSAIDs). The aim of this study was to investigate the influence of sample incubation at 37 °C for one hour on serum TXB2 concentration in comparison with incubation at room temperature. A total of 54 blood samples for serum TXB2 measurements were collected from six healthy beagle dogs into two separate serum tubes. While one group of tubes was incubated in a 37 °C water bath, the second group of tubes was left to coagulate at room temperature, both for one hour. Serum TXB2 concentrations were measured by ELISA. The mean concentration (± SD) of serum TXB2 in the group of samples that were incubated at 37 °C was significantly (P < 0.0001) higher compared to the group of samples incubated at room temperature, 1098 ± 346 μg/l and 550 ± 257 μg/l, respectively. The results of the study provide the information on serum TXB2 concentration in healthy beagle dogs and demonstrate that validated methods for assessment of COX-1 activity by measurement of serum TXB2 should be used in order to make results more reliable and comparable between different studies. The results of this study might be of great help in planning NSAID studies in dogs by providing the information that TXB2 generation by platelets is influenced profoundly by incubation temperature.

Voriconazole Increases while Itraconazole Decreases Plasma Meloxicam Concentrations

This study investigated the effect of voriconazole, an inhibitor of cytochrome P450 2C9 (CYP2C9) and CYP3A4, and itraconazole, an inhibitor of CYP3A4, on the pharmacokinetics and pharmacodynamics of meloxicam. Twelve healthy volunteers in a crossover study ingested 15 mg of meloxicam without pretreatment (control), after voriconazole pretreatment, and after itraconazole pretreatment. The plasma concentrations of meloxicam, voriconazole, itraconazole, and thromboxane B(2) (TxB(2)) generation were monitored. Compared to the control phase, voriconazole increased the mean area under the plasma concentration-time curve from 0 to 72 h (AUC(0-72)) of meloxicam by 47% (P < 0.001) and prolonged its mean half-life (t(1/2)) by 51% (P < 0.01), without affecting its mean peak concentration (C(max)). In contrast, itraconazole decreased the mean AUC(0-72) and C(max) of meloxicam by 37% (P < 0.001) and by 64% (P < 0.001), respectively, and prolonged its t(1/2) and time to C(max). The plasma protein unbound fraction of meloxicam was unchanged by voriconazole and itraconazole. Lowered plasma meloxicam concentrations during the itraconazole phase were associated with decreased pharmacodymic effects of meloxicam, as observed by weaker inhibition of TxB(2) synthesis compared to the control and voriconazole phases. Voriconazole increases plasma concentrations of meloxicam, whereas itraconazole, unexpectedly, decreases plasma meloxicam concentrations, possibly by impairing its absorption.

Antineuroinflammatory polyketide plakortide N from the Brazilian marine sponge Plakortis angulospiculatus

Involvement of brain microglia (BMΦ) in neuroinflammation has been hypothesized (Mayer AMS, MEDICINA 58:377, 1998; Mayer AMS et al., SHOCK 11: 180, 1999). We have reported marine chemicals as a potential source of antineuroinflammatory compounds (Mayer et al. BioMedCentral Pharmacology 5:6, 2005) using Escherichia coli lipopolysaccharide (LPS)‐activated BMΦ as our in vitro model (Mayer AMS et al., SHOCK 11:180, 1999). The purpose of this investigation was to study the effect of several polyketides isolated from the marine sponge Plakortis angulospiculatus on phorbol ester (PMA)‐stimulated thromboxane B2 (TXB2) and superoxide anion (O2−) generation from E. coli LPS‐activated rat BMΦ O2− was determined by superoxide dismutase‐inhibitable reduction of ferricytochrome C and TXB2 using commercially available immunoassays. Only plakortide N potently inhibited TXB2 (IC50=0.93μM) but not O2− (IC50 &gt;10μM), with low lactic dehydrogenase release (LDH50&gt;10μM). Because LDH release was low, our current data suggests that the significant inhibitory effect of plakortide N on BMΦ TXB2 release was pharmacologic rather than toxic in nature, although the molecular mechanism of inhibition remains undetermined at this time. (Supported by American Society of Pharmacognosy 1998 Research Starter Grant, NIH grant CA 67786 and FAPESP grants 01/03095‐0 and 05/60175‐2 to RGB and Midwestern University to AMM).TOTAL

Alteration of release and role of adenosine diphosphate and thromboxane A2 during collagen-induced aggregation of platelets from cattle with Chediak-Higashi syndrome

To compare the interaction of endogenous ADP with collagen and thromboxane A(2) (TXA(2)) during collagen-induced platelet aggregation between platelets from healthy cattle and those with Chediak-Higashi syndrome (CHS). POPULATION SAMPLE: Platelets harvested from blood samples from healthy Japanese Black cattle and those with CHS. Aggregation of gel-filtered platelets; release of ATP-ADP; and generation of thromboxane B(2) (TXB(2)), a metabolite of TXA(2), were measured. The potency of collagen to induce aggregation in platelets of cattle with CHS (ie, CHS platelets) was less than a tenth of that in platelets of healthy cattle (ie, control platelets). Platelet aggregation induced by collagen at an intermediate concentration depended on the coexistence of ADP and TXA(2), suggesting that released ADP cannot cause platelet aggregation by itself. Collagen-induced ADP release was markedly decreased, whereas TXB(2) production was slightly low in CHS platelets, compared with that in control platelets. A combination of subthreshold amounts of ADP and 9,11-dideoxy-9alpha, 11alpha-methano-epoxy-prostaglandin F(2) (U46619), a TXA(2) analogue, caused platelet aggregation. Similarly, a combination of subthreshold amounts of collagen and ADP caused platelet aggregation, whereas collagen and U46619 were not synergistic. Deficient ADP release ensuing from the delta-storage pool deficiency in platelets from cattle with CHS resulted in reduction of collagen-induced platelet aggregation, through attenuation of synergism between TXA(2) and ADP and between ADP and collagen. Furthermore, results of the study reported here indicated that TXA(2) was important for aggregation of bovine platelets.

Atrovirinone inhibits pro‐inflammatory mediator release from murine macrophages and human whole blood

Many plant-derived natural compounds have been reported previously to inhibit the production of important pro-inflammatory mediators such as nitric oxide, prostaglandin E2, TNF-alpha and reactive oxygen species by suppressing inducible enzyme expression via inhibition of the mitogen-activated protein kinase pathway and nuclear translocation of critical transcription factors. This study evaluates the effects of atrovirinone [2-(1-methoxycarbonyl-4,6-dihydroxyphenoxy)-3-methoxy-5,6-di-(3-methyl-2-butenyl)-1,4-benzoquinone)], a benzoquinone that we have previously isolated from Garcinia atroviridis, on two cellular systems that are repeatedly used in the analysis of anti-inflammatory bioactive compounds, namely, RAW 264.7 macrophage cells and whole blood. Atrovirinone inhibited the production of both nitric oxide and prostaglandin E2 from LPS-induced and IFN-gamma-induced RAW 264.7 cells and whole blood, with inhibitory concentration (IC)50 values of 4.62 +/- 0.65 and 9.33 +/- 1.47 micromol/L, respectively. Analysis of thromboxane B2 (TXB2) secretion from whole blood stimulated by either the cyclooxygenase (COX)-1 or the COX-2 pathway showed that atrovirinone inhibits the generation of TXB2 by both pathways, with IC50 values of 7.41 +/- 0.92 and 2.10 +/- 0.48 micromol/L, respectively. Analysis of IC50 ratios showed that atrovirinone was more COX-2 selective in its inhibition of TXB2, with a ratio of 0.32. Atrovirinone also inhibited the generation of intracellular reactive oxygen species and the secretion of TNF-alpha from RAW 264.7 cells in a dose-responsive manner, with IC50 values of 5.99 +/- 0.62 and 11.56 +/- 0.04 micromol/L, respectively. Lipoxygenase activity was also moderately inhibited by atrovirinone. Our results suggest that atrovirinone acts on important pro-inflammatory mediators possibly by the inhibition of the nuclear factor-kappaB pathway and also by the inhibition of the COX/lipoxygenase enzyme activity.

Differential modulation of microglia superoxide anion and thromboxane B2 generation by the marine manzamines.

BackgroundThromboxane B2 (TXB2) and superoxide anion (O2-) are neuroinflammatory mediators that appear to be involved in the pathogenesis of several neurodegenerative diseases. Because activated-microglia are the main source of TXB2 and O2- in these disorders, modulation of their synthesis has been hypothesized as a potential therapeutic approach for neuroinflammatory disorders. Marine natural products have become a source of novel agents that modulate eicosanoids and O2- generation from activated murine and human leukocytes. With the exception of manzamine C, all other manzamines tested are characterized by a complex pentacyclic diamine linked to C-1 of the β-carboline moiety. These marine-derived alkaloids have been reported to possess a diverse range of bioactivities including anticancer, immunostimulatory, insecticidal, antibacterial, antimalarial and antituberculosis activities. The purpose of this investigation was to conduct a structure-activity relationship study with manzamines (MZ) A, B, C, D, E and F on agonist-stimulated release of TXB2 and O2- from E. coli LPS-activated rat neonatal microglia in vitro.ResultsThe manzamines differentially attenuated PMA (phorbol 12-myristate 13-acetate)-stimulated TXB2 generation in the following order of decreasing potency: MZA (IC50 <0.016 μM) >MZD (IC50 = 0.23 μM) >MZB (IC50 = 1.6 μM) >MZC (IC50 = 2.98 μM) >MZE and F (IC50 >10 μM). In contrast, there was less effect on OPZ (opsonized zymosan)-stimulated TXB2 generation: MZB (IC50 = 1.44 μM) >MZA (IC50 = 3.16 μM) >MZC (IC50 = 3.34 μM) >MZD, MZE and MZF (IC50 >10 μM). Similarly, PMA-stimulated O2- generation was affected differentially as follows: MZD (apparent IC50<0.1 μM) >MZA (IC50 = 0.1 μM) >MZB (IC50 = 3.16 μM) >MZC (IC50 = 3.43 μM) >MZE and MZF (IC50 >10 μM). In contrast, OPZ-stimulated O2- generation was minimally affected: MZB (IC50 = 4.17 μM) >MZC (IC50 = 9.3 μM) >MZA, MZD, MZE and MZF (IC50 > 10 μM). From the structure-activity relationship perspective, contributing factors to the observed differential bioactivity on TXB2 and O2- generation are the solubility or ionic forms of MZA and D as well as changes such as saturation or oxidation of the β carboline or 8-membered amine ring. In contrast, the fused 13-membered macrocyclic and isoquinoline ring system, and any substitutions in these rings would not appear to be factors contributing to bioactivity.ConclusionTo our knowledge, this is the first experimental study that demonstrates that MZA, at in vitro concentrations that are non toxic to E. coli LPS-activated rat neonatal microglia, potently modulates PMA-stimulated TXB2 and O2- generation. MZA may thus be a lead candidate for the development of novel therapeutic agents for the modulation of TXB2 and O2- release in neuroinflammatory diseases. Marine natural products provide a novel and rich source of chemical diversity that can contribute to the design and development of new and potentially useful anti-inflammatory agents to treat neurodegenerative diseases.

Plättchenfunktionstests zum Monitoring der Azetylsalizylsäuretherapie

ZusammenfassungMehrere Studien mit verschiedenen Plättchenfunktionstests (PFT) zeigten, dass Subgruppen von Patienten unter Azetylsalizylsäure(ASS)-Therapie den erwarteten plättchenfunktionshemmenden Effekt vermissen lassen. Dieses Phänomen wird ebenso wie das klinische Versagen einer ASS-Prophylaxe als ASS-Resistenz (AR) oder Nichtansprechen (nonresponsiveness) auf ASS bezeichnet. Mithilfe von PFT lassen sich mehrere Subtypen unterscheiden. Zur Charakterisierung einer AR wurden bislang folgende PFT herangezogen: optische Aggregometrie, Vollblut-Aggregometrie, Plättchenfunktionsanalyzer PFA-100, Plättchenreaktivitätsindex, Durchflusszytometrie und die Ausscheidung von Metaboliten des Thromboxans B2 im Urin oder die Thromboxan-B2-Generation in plättchenreichem Plasma. Eine komplizierte Präanalytik und Analytik sowie unspezifische Störeinflüsse und schlechte Reproduzierbarkeit schränken ihre Anwendung in der klinischen Routine erheblich ein. Darüber hinaus sind unterschiedliche PFT offenbar nicht austauschbar im Hinblick auf die Beurteilung einer AR. Drei prospektive klinische Studien wiesen eine Assoziation zwischen einer vermeintlichen, laboranalytisch festgestellten AR und kardiovaskulären Ereignissen nach. Daher besteht dringender Bedarf an einem einfachen und reproduzierbaren Test, der zuverlässig die individuelle klinische Wirksamkeit einer Therapie mit Plättchenfunktionshemmern anzeigt. Keiner der aktuell verfügbaren PFT einschließlich des PFA-100-Systems wird gegenwärtig diesem Anspruch gerecht.

Nitric oxide-releasing NSAIDs: a review of their current status.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most widely prescribed drugs worldwide owing to their anti-inflammatory, antipyretic and analgesic properties. However, their use is hampered by gastrointestinal (GI) toxicity, the most common drug-related serious adverse event in industrialised nations. Nitric oxide (NO)-releasing NSAIDs, a recently described class of drugs, are generated by adding a nitroxybutyl or a nitrosothiol moiety to the parent NSAID via a short-chain ester linkage. While efficacy of nitrosothiol-NO-NSAIDs still awaits investigation, nitroxybutyl-NO-NSAIDs have been extensively studied in animals, thus the abbreviation NO-NSAIDs used here refers to the latter group of NSAID derivatives. NO-NSAIDs retain the anti-inflammatory and antipyretic activity of original NSAIDs, although they exhibit markedly reduced gastrointestinal toxicity. NO-NSAIDs are nonselective cyclo-oxygenase (COX) inhibitors, and they also exert COX-independent activities that are NO-dependent. Indeed, NO-NSAIDs suppress production of the cytokines interleukin (IL)-1beta, IL-18 and interferon-gamma by causing the S-nitrosilation/inhibition of caspase-1. In acute and chronic animal models of inflammation, it has been demonstrated that NO-NSAIDs abrogated prostaglandin E2 as well as thromboxane B2 generation. In a murine model, NO-naproxen was approximately 10-fold more potent than naproxen in reducing animal writhing after intraperitoneal injection of acetic acid. Similar data have been obtained in chronic models of pain such as rat adjuvant arthritis. In vivo and in vitro studies suggest that NO-aspirin (acetylsalicylic acid) exerts more potent antithrombotic action than aspirin, probably by coupling the ability to inhibit COX-1 with the anti-adhesive effect of NO. Moreover, in a model of renal injury NO-flurbiprofen not only has been demonstrated to be devoid of nephrotoxicity but also to ameliorate renal function. Finally, in an animal model of chronic neurodegenerative disease, NO-flurbiprofen and NO-aspirin attenuated the brain inflammatory response. The GI toxicity of NO-flurbiprofen and NO-naproxen is currently being investigated in healthy individuals.

Effect of flosulide, a selective cyclooxygenase 2 inhibitor, on passive Heymann nephritis in the rat

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce an inhibition of cyclooxygenase (COX), an enzyme that makes prostaglandins. Two isoforms of COX exist: COX-1 represents the constitutively expressed enzyme, whereas COX-2 is the inducible isoform. This study investigated the role of COX-2 in the inflammatory processes of the kidneys of rats with passive Heymann nephritis (PHN), and focused of the effect of a selective COX-2 inhibitor, flosulide. COX-2-selective inhibitors are thought to represent potent anti-inflammatory agents without major renal side effects. PHN was induced by injecting heterologous Fx1A antiserum into female Wistar rats. Two treatment groups, each consisting of 12 rats with PHN, received either 3 or 9 mg of flosulide/kg body wt/day and were compared with untreated controls. After four weeks, the generation of thromboxane B2 (TxB2) and 6-keto-PGF1alpha were determined in renal tissue and in urine. COX-2 protein expression was investigated by Western blotting using a selective antibody. Rats with PHN exhibited a marked proteinuria of 71 +/- 8 mg/24 hr as compared with 2.0 +/- 0.3 mg/24 hr in healthy controls (P < 0.01). Treatment with flosulide reduced the proteinuria to 26.1 +/- 9 mg/24 hr at 3 mg flosulide/kg body wt/day and 35.5 +/- 6 mg/24 hr at 9 mg/kg body wt/day, which was equivalent to a reduction of proteinuria by a maximum of 65% (P < 0.05). This was accompanied by an increase in glomerular TxB2 from 3073 +/- 355 to 5255 +/- 1041 pg/mg glomerular protein and 6-keto-PGF1alpha from 1702 +/- 161 to 2724 +/- 770 pg/mg glomerular protein in rats with PHN. COX-2 protein expression was also highly elevated in comparison to healthy controls. Low-dose flosulide treatment had no effect on COX protein expression and renal prostaglandin formation. High-dose flosulide treatment reduced renal prostaglandin production and caused a marked decline in COX-1 and COX-2 protein expression. Urine prostanoid excretion remained unchanged in all therapeutic groups. There was a small though significant reduction in renal creatinine clearance from 0.86 ml +/- 0.2/min in untreated controls to 0.6 ml +/- 0.1/min in flosulide-treated rats with PHN (P < 0.01) after four weeks. Under the influence of flosulide, a highly COX-2-selective inhibitor, we observed an antiproteinuric drug effect. The inflammation in PHN induced COX-2 protein expression that was not affected by low-dose flosulide. COX-1 and COX-2 protein expression was affected by high-dose flosulide, which therefore might lose its selectivity. High-dose flosulide induced a decrease in glomerular prostanoid production possibly because of COX-1 inhibition. Our results suggest that the therapeutic use of flosulide in proteinuria seems advantageous and deserves further studies because the basal prostaglandin levels remain unchanged in the low-dose-treated group, indicating that the compensatory capacity of prostaglandin production, which is essential for the regulation of renal hemodynamics, is maintained.

Inhibition of leukocyte eicosanoid generation and radical scavenging activity by gnaphalin, a lipophilic flavonol isolated from Helichrysum picardii.

The lipophilic aglycone 5,7-dihydroxy-3,8-dimethoxyflavone (gnaphalin) isolated from the aerial flowering parts of Helichrysum picardii Boiss. & Reuter (Asteraceae) was tested for interactions with the cyclo-oxygenase and 5-lipoxygenase pathways of arachidonate metabolism in stimulated rat peritoneal leukocytes, and for its effects on leukocyte granular enzyme release, cell viability and interactions with reactive oxygen species. Gnaphalin dose-dependently inhibited generation of the cyclo-oxygenase metabolite thromboxane B2 (IC50 = 39.9 +/- 3.9 microM), and of the 5-lipoxygenase metabolite leukotriene B4, although the potency was two-fold less (IC50 = 81.8 +/- 12.9 microM). At concentrations of 6 to 320 microM, gnaphalin did not affect secretion of the pro-inflammatory enzymes lysozyme, myeloperoxidase and beta-glucuronidase from the neutrophil secretory granules, and did not scavenge hydrogen peroxide or hypochlorous acid. However, gnaphalin effectively scavenged superoxide radicals generated in the hypoxanthine/xanthine oxidase system (IC50 = 40 microM) and by PMA-stimulated leukocytes (> 60% at 500 microM), directly inhibited xanthine oxidase (85% at 395 microM) and inhibited Fe(3+)-ascorbate-induced liposomal peroxidation (IC50 = 215 microM). Thus, like some other flavonoids found in medicinal herbs, gnaphalin possesses an array of potentially beneficial anti-eicosanoid and free-radical scavenging properties which may alongside other constituents contribute to the claimed medicinal properties of the plant from which it is derived.

YM337, A platelet glycoprotein IIb/IIIa antagonist, lessens photochemically-induced ischemic brain damage in monkeys

To elucidate the role of platelets in brain damage during focal cerebral ischemia, the effect of YM337, a glycoprotein IIb/IIIa antagonist, on infarct areas and neurologic deficits was measured in monkeys with thrombotic distal middle cerebral artery occlusions. Distal middle cerebral arteries were occluded by a platelet-rich thrombus formed after photochemical reaction between rose bengal dye and green light. The experimental drugs were intravenously injected 30 min after rose bengal injection and continuously infused for 24 h thereafter. YM337, but not the thromboxane A2 synthetase inhibitor sodium ozagrel, significantly inhibited ex vivo ADP-induced platelet aggregation. The percentage of ADP-induced platelet aggregation 4 h after the start of administration was 87.0% of predosing value in the saline group, 55.6% in the YM337 low dose group, 28.8% in the YM337 high dose group and 89.0% in the sodium ozagrel group. However, while sodium ozagrel significantly inhibited thromboxane B2 generation accompanying arachidonic acid-induced platelet aggregation, YM337 had little effect on this pathway. The neurologic deficit was milder and infarct area significantly smaller in the YM337-treated groups compared with the saline control group. The ratio of infarcted area to the whole area of the cerebrum was 12.3% in the saline group, 5.5% in the YM337 low dose group, 5.7% in the YM337 high dose group, and 11.0% in the ozagrel group. These results suggest that a blockade of glycoprotein IIb/IIIa is a beneficial approach to treat cerebral artery thrombosis and cerebral infarction. Drug Dev. Res. 45:162–169, 1999. © 1999 Wiley-Liss, Inc.

Restoration of the disordered glucose-fatty acid cycle in alloxan-diabetic rats by trihydroxyoctadecadienoic acids from Bryonia alba, a native Armenian medicinal plant.

We studied how administration of trihydroxyoctadecadienoic acids obtained from the roots of the native Armenian plant Bryonia alba L. (0.05 mg/kg/day for 15 days. i.m.) restores the disordered lipid metabolism of alloxan-diabetic rats. Diabetes was accompanied by an increase in total non-esterified fatty acid content of blood together with decreases in muscle and adipose tissue of total non-esterified fatty acids and triglycerides, together with marked alterations of phospholipid fatty acid distribution within the membranes from muscle, including increased short chain fatty acid and decreased arachidonate content. All these metabolic changes induced in diabetes were significantly restored by trihydroxyoctadecadienoic acid treatment towards their normal values (P < 0.005) with the exception of diminished triglyceride content of muscle which was not restored. In experiments on rat neutrophil leukocytes in vitro, it was found that the standardised preparation of Bryonia C-18 fatty acids (a mixture of four diastereoisomeric forms of the positional isomers I 12,13,16-trihydroxy-9Z,14E-octadecadienoic acid, II 12,15,16-trihydroxy-9Z,13E-octadecadienoic acid, III 9,10, 13-trihydroxy-11E,15Z-octadecadienoic acid and IV 9,12,13-trihydroxy-10E,14Z-octadecadienoic acid) had no effect on granular enzyme secretion or 5-lipoxygenase activity at 5-50 micrograms/ml, but dose-dependently reduced thromboxane B2 generation, with a corresponding increase in prostaglandin E2 release. We conclude that trihydroxyoctadecadienoic acids from B. alba can correct major metabolic abnormalities typical of severe diabetes mellitus, and that they can influence the profile of the formation of stable prostaglandins by actions downstream of prostaglandin endoperoxides.

Effects of ibuprofen enantiomers and its coenzyme A thioesters on human prostaglandin endoperoxide synthases.

1. Ibuprofen enantiomers and their respective coenzyme A thioesters were tested in human platelets and blood monocytes to determine their selectivity and potency as inhibitors of cyclo-oxygenase activity of prostaglandin endoperoxide synthase-1 (PGHS-1) and PGHS-2. 2. Human blood from volunteers was drawn and allowed to clot at 37 degrees C for 1 h in the presence of increasing concentrations of the test compounds (R-ibuprofen, S-ibuprofen, R-ibuprofenoyl-CoA, S-ibuprofenoyl-CoA, NS-398). Immunoreactive (ir) thromboxane B2 (TXB2) concentrations in serum were determined by a specific EIA assay as an index of the cyclo-oxygenase activity of platelet PGHS-1. 3. Heparin-treated blood from the same donors was incubated at 37 degrees C for 24 h with the same concentrations of the test compounds in the presence of lipopolysaccharide (LPS, 10 microg ml[-1]). The contribution of PGHS-1 was suppressed by pretreatment of the volunteers with aspirin (500 mg; 48 h before venepuncture). As a measure of LPS induced PGHS-2 activity immunoreactive prostaglandin E2 (irPGE2) plasma concentrations were determined by a specific EIA assay. 4. S-ibuprofen inhibited the activity of PGHS-1 (IC50 2.1 microM) and PGHS-2 (IC50 1.6 microM) equally. R-ibuprofen inhibited PGHS-1 (IC50 34.9) less potently than S-ibuprofen and showed no inhibition of PGHS-2 up to 250 microM. By contrast R-ibuprofenoyl-CoA thioester inhibited PGE2 production from LPS-stimulated monocytes almost two orders of magnitude more potently than the generation of TXB2 (IC50 5.6 vs 219 microM). 5. Western blotting of PGHS-2 after LPS induction of blood monocytes showed a concentration-dependent inhibition of PGHS-2 protein expression by ibuprofenoyl-CoA thioesters. 6. These data confirm that S-ibuprofen represents the active entity in the racemate with respect to cyclo-oxygenase activity. More importantly the data suggest a contribution of the R-enantiomer to therapeutic effects not only by chiral inversion to S-ibuprofen but also via inhibition of induction of PGHS-2 mediated by R-ibuprofenoyl-CoA thioester. 7. The data may explain why racemic ibuprofen is ranked as one of the safest non-steroidal anti-inflammatory drugs (NSAIDs) so far determined in epidemiological studies.

Lipid peroxide overcomes the inability of platelet secretory phospholipase A2 to hydrolyze membrane phospholipids in rabbit platelets.

The present study investigated the effect of lipid peroxide on the ability of group IIA secretory phospholipase A2 (IIAsPLA2) to hydrolyze platelet membrane phospholipids. The treatment of rabbit platelets with tert-butyl hydroperoxide (BHP) and FeSO4 generated malondialdehyde, an index of lipid peroxidation, and slightly induced arachidonic acid liberation and lysophosphatidylcholine formation. Further addition of IIAsPLA2 purified from rabbit platelets synergistically enhanced the liberation and the formation induced by the oxidizing reagents, although the enzyme alone did not. When the IIAsPLA2 was pretreated with heparin, the enhancement was not observed. The combination of IIAsPLA2 with linoleic acid hydroperoxide and FeSO4 also caused synergistic arachidonic acid liberation. Furthermore, IIAsPLA2 enhanced thromboxane B2 generation and platelet aggregation induced by BHP and FeSO4. The synergistic aggregation was sensitive to indomethacin. With a membrane fraction as a substrate, IIAsPLA2 caused arachidonic acid liberation, which was enhanced in the presence of BHP and FeSO4. These results suggest that modification of membrane phospholipids by oxidizing reagents increases the accessibility of the membrane to platelet IIAsPLA2, and sequential enhancement of arachidonic acid liberation may contribute to the propagation of oxidative stress-induced cellular injury.

Sulphydryl blocker induced small intestinal inflammation in rats: a new model mimicking Crohn’s disease

Background—Sulphydryl compounds are essential for maintaining mucosal integrity in the gastrointestinal tract.Aim—To characterise a model of experimental inflammation in the small intestine induced by a sulphydryl blocker.Methods—Inflammation in the small...

Pharmacokinetic and pharmacodynamic studies on phenylbutazone and oxyphenbutazone in goats

Phenylbutazone was administered intravenously and orally to six goats as a single dose of 4.4 mg/kg and its disposition and bioavailability and the disposition of its active metabolite, oxyphenbutazone, in...

The H1-histamine antagonist dithiaden inhibits human platelet function in vitro

The H1-histamine receptor antagonist Dithaden® inhibited in a dose-dependent manner, human platelet aggregation in vitro that was induced with stimuli in the following rank order of potency: thrombin > A23187 > adrenaline > ADP. The aggregation of platelets in plasma induced with adrenaline was inhibited by Dithiaden (DIT) (both the first and the second phase) and the onset of the second phase was prolonged significantly. In a concentration-dependent manner DIT inhibited thrombin- and calcium ionophore A23187-induced [3H]arachi-donic acid liberation from, and peroxidation (measured as malondialdehyde formation) of membrane phospholipids. The same effect of DIT was found for the inhibition of thromboxane B2 generation. It is suggested that DIT does not inhibit stimulated platelets at specific histamine receptors sites but rather at the phospholipase A2 and thromboxane synthase level. Results from in vitro experiments should be verified in vivo.

Effect of manoalide on human 5-lipoxygenase activity.

The marine natural product manoalide (MLD) has been described to inactivate phospholipase A2 (PLA2) from several sources as well as to inhibit synthesis of eicosanoids in human polymorphonuclear leukocytes (HPMNL). MLD also reduces chemically-induced inflammation in vivo. In this investigation we have examined the effect of MLD on A23187-induced generation of leukotriene B4 (LTB4) and thromboxane B2 (TXB2) in HPMNL as well as 5-lipoxygenase (5-LO) activity from HPMNL sonicated preparations. In the intact cell system, MLD inhibited with similar potency biosynthesis of LTB4 and TXB2 (IC50 1.7 and 1.4 microM, respectively). In order to discern if inhibition of 5-LO is involved in the effect of MLD, we examined the action of this compound on 5-LO activity from 10,000 x g and 100,000 x g supernatants of sonicated HPMNL homogenates. The enzymatic activity was not affected at concentrations of MLD up to 50 microM. These data indicate that MLD is not a direct inhibitor of 5-LO activity from HPMNL and support the hypothesis that its anti-inflammatory action could be related with a reduction of eicosanoid biosynthesis via inhibition of PLA2.

Effect of chloroquine on arachidonic acid pathway in isolated mast cells

Chloroquine (CQ) interacts with the arachidonic acid (AA) pathway in many cells and tissues as a result of phospholipase A2 inhibition. Previously, we reported the inhibitory effect of CQ on stimulated secretion from isolated rat mast cells along with the inhibition of stimulated AA liberation and thromboxane B2 (TXB2) production from membrane phospholipids [1, 2]. In this study we investigated the effect of CQ on prostagiandin D2 (PGD2) generation (a major cyclooxygenase metabolite of arachidonic acid in mast cells) [3], in compound 48/80 stimulated isolated mast cells and evaluated the relationship between AA liberation, PGD2 and TXB2 generation from membrane phospholipids.