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1.
Nature ; 527(7579): 539-543, 2015 Nov 26.
Article in English | MEDLINE | ID: mdl-26524521

ABSTRACT

Many peroxy-containing secondary metabolites have been isolated and shown to provide beneficial effects to human health. Yet, the mechanisms of most endoperoxide biosyntheses are not well understood. Although endoperoxides have been suggested as key reaction intermediates in several cases, the only well-characterized endoperoxide biosynthetic enzyme is prostaglandin H synthase, a haem-containing enzyme. Fumitremorgin B endoperoxidase (FtmOx1) from Aspergillus fumigatus is the first reported α-ketoglutarate-dependent mononuclear non-haem iron enzyme that can catalyse an endoperoxide formation reaction. To elucidate the mechanistic details for this unique chemical transformation, we report the X-ray crystal structures of FtmOx1 and the binary complexes it forms with either the co-substrate (α-ketoglutarate) or the substrate (fumitremorgin B). Uniquely, after α-ketoglutarate has bound to the mononuclear iron centre in a bidentate fashion, the remaining open site for oxygen binding and activation is shielded from the substrate or the solvent by a tyrosine residue (Y224). Upon replacing Y224 with alanine or phenylalanine, the FtmOx1 catalysis diverts from endoperoxide formation to the more commonly observed hydroxylation. Subsequent characterizations by a combination of stopped-flow optical absorption spectroscopy and freeze-quench electron paramagnetic resonance spectroscopy support the presence of transient radical species in FtmOx1 catalysis. Our results help to unravel the novel mechanism for this endoperoxide formation reaction.


Subject(s)
Aspergillus fumigatus/enzymology , Biocatalysis , Ketoglutaric Acids/metabolism , Prostaglandin Endoperoxides/biosynthesis , Binding Sites , Crystallography, X-Ray , Electron Spin Resonance Spectroscopy , Heme , Hydroxylation , Indoles/metabolism , Iron/metabolism , Oxygen/metabolism , Tyrosine/metabolism
2.
J Biol Chem ; 287(30): 25434-9, 2012 Jul 20.
Article in English | MEDLINE | ID: mdl-22654101

ABSTRACT

T helper 2 (Th2) cells play a central role in the progression of many diseases such as allergic airway inflammation, autoimmune diseases, and infections caused by intracellular pathogens. Consequently, animals such as BALB/c mice, which exhibit a propensity for generating Th2 responses, are susceptible to allergic airway inflammation, type-II autoimmune diseases, and various infections induced by intracellular pathogens, namely, Leishmania. In contrast, C3H/OuJ mice have a tendency for generating T helper 1 (Th1) responses and show resistance to these diseases. Here, we show that prostaglandin endoperoxide E(2) selectively inhibits activation-induced cell death of Th2 cells by signaling through its receptor E-prostanoid receptor 2 (EP2). Consequently, Th2 cells derived from BALB/c mice expressed very high levels of EP2. On the other hand, Th2 cells derived from C3H/OuJ mice expressed very low levels of EP2, which failed to support the survival of Th2 cells. Furthermore, we found that this effect of EP2 on Th2 cells from BALB/c mice was executed by a granzyme B-mediated mechanism. EP2 belongs to a group of G-protein-coupled receptors that are amenable to therapeutic targeting. Our findings therefore identify EP2 as a promising target for small molecule-directed immunomodulation.


Subject(s)
Lymphocyte Activation , Receptors, Prostaglandin E, EP2 Subtype/immunology , Signal Transduction/immunology , Th2 Cells/immunology , Animals , Cell Death/genetics , Cell Death/immunology , Cell Survival/genetics , Cell Survival/immunology , Dinoprostone/genetics , Dinoprostone/metabolism , Female , Mice , Mice, Inbred BALB C , Prostaglandin Endoperoxides/genetics , Prostaglandin Endoperoxides/immunology , Receptors, Prostaglandin E, EP2 Subtype/genetics , Signal Transduction/genetics
3.
Vestn Ross Akad Med Nauk ; (4): 22-6, 2010.
Article in Russian | MEDLINE | ID: mdl-20540350

ABSTRACT

The role of metabolic products of arachidonic acid and thromboxans in metabolic syndrome was evaluated in 42 patients and 16 healthy subjects. The levels of arachidonic acid and thromboxane were shown to be elevated in patients with metabolic syndrome which accounted for enhanced platelet aggregation in response to ADP, adrenaline, and collagen. It is concluded that that decreased level of cyclic nucleotides (cAMP) and prostacyclin in combination with a rise in the content of Willebrand factor in patients with metabolic syndrome is a major contributor to the development of platelet activity.


Subject(s)
Metabolic Syndrome/blood , Platelet Aggregation , Prostaglandins/blood , Adenosine Diphosphate/pharmacology , Arachidonic Acid/blood , Blood Platelets/drug effects , Blood Platelets/physiology , Collagen/pharmacology , Cyclic AMP/blood , Epinephrine/pharmacology , Epoprostenol/blood , Hemostasis , Humans , In Vitro Techniques , Prostaglandin Endoperoxides/blood , Thromboxanes/blood , von Willebrand Factor/analysis
4.
Bioorg Med Chem Lett ; 20(1): 338-40, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-19914067

ABSTRACT

The reaction pathways of deprotonation versus nucleophilic substitution involving mPGES-2 enzyme catalysis were investigated by ab initio molecular orbital theory calculations for the reaction of methylthiolate with the endoperoxide core of PGH(2) and by the combined quantum mechanical molecular mechanical methods. The calculations showed that deprotonation mechanism is energetically more favorable than the nucleophilic substitution pathway.


Subject(s)
Intramolecular Oxidoreductases/metabolism , Prostaglandin Endoperoxides/chemistry , Binding Sites , Catalysis , Computer Simulation , Models, Molecular , Prostaglandin-E Synthases
5.
Appl. cancer res ; 28(3): 83-84, July-Dec. 2008.
Article in English | LILACS, Inca | ID: lil-512883
6.
Org Biomol Chem ; 6(18): 3354-61, 2008 Sep 21.
Article in English | MEDLINE | ID: mdl-18802642

ABSTRACT

The model prostaglandin endoperoxide, 1,4-diphenyl-2,3-dioxabicyclo[2.2.1]heptane (3), was investigated in N,N-dimethylformamide at a glassy carbon electrode using various electrochemical techniques. Reduction of 3 occurs by a concerted dissociative electron transfer (ET) mechanism. Electrolysis at -1.6 V yields 1,3-diphenyl-cyclopentane-cis-1,3-diol in 97% by a two-electron mechanism; however, in competition with the second ET from the electrode, the resulting distonic radical-anion intermediate undergoes a beta-scission fragmentation. The rate constant for the heterogeneous ET to the distonic radical-anion is estimated to occur on the order of 2 x 10(7) s(-1). In contrast, electrolyses conducted at potentials more negative than -2.1 V yield a mixture of primary and secondary electrolysis products including 1,3-diphenyl-cyclopentane-cis-1,3-diol, 1,3-diphenyl-1,3-propanedione, trans-chalcone and 1,3-diphenyl-1,3-hydroxypropane by a mechanism involving less than one electron equivalent. These observations are rationalized by a catalytic radical-anion chain mechanism, which is dependent on the electrode potential and the concentration of weak non-nucleophilic acid. A thermochemical cycle for calculating the driving force for beta-scission fragmentation from oxygen-centred biradicals and analogous distonic radical-anions is presented and the results of the calculations provide insight into the reactivity of prostaglandin endoperoxides.


Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/chemistry , Electrons , Prostaglandin Endoperoxides/chemical synthesis , Anions/chemistry , Kinetics , Models, Biological , Molecular Structure , Oxidation-Reduction , Prostaglandin Endoperoxides/chemistry , Thermodynamics
8.
Eur J Med Chem ; 39(5): 389-96, 2004 May.
Article in English | MEDLINE | ID: mdl-15110965

ABSTRACT

Novel 9,11-ethano analogues of prostaglandin endoperoxides with a nitrogen in position 13 were synthesized. (1)H NMR spectra of the obtained compounds were studied. All prostanoids administered perorally at doses of 2.5-10.0 microg x kg(-1) had specific dose-dependent effects on the B-cellular immunity estimated under in vivo conditions on the model of the B-cellular immune response. In terms of the direction of their activities, eight of the studied compounds were found to be immunostimulators, whereas other three compounds displayed immunosuppressing effect. Two of the compounds increased the amount of antibody-forming cells (AFC) per 10(6) spleen cells by 1.9 times in comparison with the respective parameter of control group.


Subject(s)
B-Lymphocytes/immunology , Prostanoic Acids/chemical synthesis , Prostanoic Acids/pharmacology , Animals , B-Lymphocytes/drug effects , Bridged Bicyclo Compounds/chemistry , Dose-Response Relationship, Drug , Female , Hydrogen/chemistry , Magnetic Resonance Spectroscopy/methods , Mice , Mice, Inbred CBA , Molecular Structure , Prostaglandin Endoperoxides/chemistry , Prostanoic Acids/chemistry , Spleen/cytology , Spleen/drug effects , Spleen/immunology
9.
FASEB J ; 17(8): 914-6, 2003 May.
Article in English | MEDLINE | ID: mdl-12670882

ABSTRACT

In bovine coronary artery segments, peroxynitrite inhibits prostacyclin (PGI2) synthase by tyrosine nitration. Using this pharmacological model, we show that a 1 h exposure of bovine coronary artery segments to endotoxin (lipopolysaccharide [LPS]) inhibits the relaxation phase following angiotensin II (Ang II) stimulation and causes a vasospasm that can be suppressed by a thromboxane A2 (TxA2) receptor blocker. In parallel, PGI2 synthesis decreases in favor of prostaglandin E2 formation. Immunoprecipitation and costaining with an anti-nitrotyrosine antibody identified PGI2 synthase as the main nitrated protein in the endothelium. All effects of LPS could be prevented in the presence of the nitric oxide (NO) synthase inhibitor Nomega-mono-methyl-L-arginine and polyethylene-glycolated Cu/Zn- superoxide dismutase. Thus, the early phase of endothelial cell activation in bovine coronary arteries by inflammatory agents proceeds by a protein synthesis-independent priming process for a source of superoxide that we tentatively attribute to xanthine oxidase. Upon receptor activation, Ang II stimulates NO and superoxide production, resulting in a peroxynitrite-mediated nitration and inhibition of PGI2 synthase. The remaining 15-hydroxy-prostaglandin 9,11-endoperoxide (PGH2) first activates the TxA2/PGH2 receptor and then is converted to prostaglandin E2 (PGE2) by smooth muscle cells. PGE2 together with a lack of NO and PGI2 is known to promote the adhesion of white blood cells and their immigration to the inflammatory locus.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Endothelium, Vascular/drug effects , Intramolecular Oxidoreductases/metabolism , Lipopolysaccharides/pharmacology , Nitric Oxide/metabolism , Receptors, Thromboxane/metabolism , Superoxides/metabolism , 6-Ketoprostaglandin F1 alpha/metabolism , Angiotensin II/pharmacology , Animals , Cattle , Coronary Vasospasm/chemically induced , Coronary Vasospasm/metabolism , Coronary Vessels/drug effects , Coronary Vessels/metabolism , Coronary Vessels/physiology , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , In Vitro Techniques , Nitrates/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Peroxynitrous Acid/metabolism , Polyethylene Glycols/pharmacology , Prostaglandin Endoperoxides/metabolism , Superoxide Dismutase/pharmacology , Vasoconstriction/drug effects , Vasodilation/drug effects , Xanthine Oxidase/metabolism , omega-N-Methylarginine/pharmacology
10.
J Cardiovasc Pharmacol ; 40(6): 930-7, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12451327

ABSTRACT

The current investigation explored the potential age-dependant modulation of abnormal spontaneous constrictions (thromboxane-like) in the spontaneously hypertensive rat (SHR) aorta, observed only after the inhibition of endogenous production of nitric oxide (NO). Aortic rings from SHR and Wistar-Kyoto (WKY) control rats of varying ages (4, 8, 12, and 18 months) were mounted in organ baths, and changes in tension were monitored. Inhibition of NO with Nomega-nitro-L-arginine (NOLA) unmasked a slow contraction, which appeared to be age dependent (p < 0.05). This contraction was found in SHRs of all age groups and in older WKY rats. Denuding the endothelium in young SHRs did not influence the constriction, confirming a nonendothelial cell origin, while in the older groups this led to a 30-40% reduction in contraction. Comparable attenuation of the constrictor response was observed after incubation of endothelium intact rings with superoxide dismutase (100 U/ml) or 3-amino-1,2,4-triazole. Of the residual activity that was unaffected by free radical scavengers or de-endothelialization, 60-70% was sensitive to cyclooxygenase inhibition by indomethacin and/or ibuprofen. The thromboxane (TxA ) receptor antagonist SQ29548 induced a complete reversal of the abnormal constriction. In contrast, thromboxane synthetase inhibition had no effect, ruling out any involvement of TxA in mediating this abnormality. Collectively, these observations support the view that as compared with the normotensive setting, contraction induced by NO inhibition in the SHR develops prematurely and deteriorates more rapidly during the aging process. In aged rats, prostaglandin endoperoxide intermediates PGG /H and endothelium-derived free radicals rather than TxA per se appear to contribute to the NOLA-dependent TxA -like vasoconstriction.


Subject(s)
Aging/physiology , Hypertension/physiopathology , Muscle, Smooth, Vascular/drug effects , Vasoconstriction/drug effects , Animals , Aorta/drug effects , Drug Interactions , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Male , Prostaglandin Endoperoxides/physiology , Rats , Rats, Inbred SHR , Rats, Inbred WKY
11.
Endocrinology ; 142(7): 3187-97, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11416041

ABSTRACT

Normal ovulation in mice requires PG-endoperoxide synthase 2 (cyclooxygenase-2; COX-2) expression. This study examined the role of the oocyte and other factors in regulating steady state levels of COX-2 messenger RNA (mRNA) in granulosa cells. Multiphasic changes in the expression pattern of COX-2 mRNA were found, with peaks of expression 4 and 12 h after hCG treatment. Changes in relative expression levels in cumulus cells and mural granulosa cells occurred over time, with similar mRNA levels at 4 h, but higher levels in cumulus cells compared with mural granulosa cells at 8 and 12 h post-hCG. In cultured mural granulosa cells, LH, FSH, and oocytes promoted COX-2 mRNA expression concurrent with the first expression peak in vivo. At the same time, FSH, but not LH, treatment of cultured cumulus-oocyte complexes (COC) promoted COX-2 mRNA expression in cumulus cells. This response of cumulus cells to FSH treatment was largely dependent on the presence of either fully grown germinal vesicle stage or maturing oocytes, but not growing oocytes. At 8 h, COX-2 mRNA expression in FSH-stimulated COC was lower than at 4 h; however, oocyte coculture promoted COX-2 mRNA expression in cumulus cells. No second peak in expression occurred in cultured COC. However, coculture of COC with follicle walls promoted COX-2 mRNA expression in cumulus cells 12 h post-hCG; an effect augmented by oocytes. Therefore, the oocyte resident within ovulatory follicles produces a factor(s) that promotes expression of COX-2 mRNA by cumulus cells and possibly by mural granulosa cells. Thus, the oocyte probably plays an important role in promoting ovulation. However, the multiphasic changes in the pattern of COX-2 expression appear orchestrated by non-oocyte-derived factors.


Subject(s)
Granulosa Cells/metabolism , Ovulation/physiology , Prostaglandin Endoperoxides/genetics , RNA, Messenger/metabolism , Animals , Cells, Cultured , Chorionic Gonadotropin/pharmacology , Cyclooxygenase 2 , Female , Gene Expression Regulation/physiology , Homeostasis , Isoenzymes/genetics , Kinetics , Mice , Oocytes/physiology , Ovarian Follicle/cytology , Ovarian Follicle/drug effects , Ovarian Follicle/metabolism , Prostaglandin-Endoperoxide Synthases/genetics , Time Factors
12.
J Biol Chem ; 275(29): 21844-9, 2000 Jul 21.
Article in English | MEDLINE | ID: mdl-10791960

ABSTRACT

A novel cytochrome P450, CYP4F8, was recently cloned from human seminal vesicles. CYP4F8 was expressed in yeast. Recombinant CYP4F8 oxygenated arachidonic acid to (18R)-hydroxyarachidonate, whereas prostaglandin (PG) D(2), PGE(1), PGE(2), PGF(2alpha), and leukotriene B(4) appeared to be poor substrates. Three stable PGH(2) analogues, 9,11-epoxymethano-PGH(2) (U-44069), 11, 9-epoxymethano-PGH(2) (U-46619), and 9,11-diazo-15-deoxy-PGH(2) (U-51605) were rapidly metabolized by omega2- and omega3-hydroxylation. U-44069 was oxygenated with a V(max) of approximately 260 pmol min(-)(1) pmol P450(-1) and a K(m) of approximately 7 micrometer. PGH(2) decomposes mainly to PGE(2) in buffer and to PGF(2alpha) by reduction with SnCl(2). CYP4F8 metabolized PGH(2) to 19-hydroxy-PGH(2), which decomposed to 19-hydroxy-PGE(2) in buffer and could be reduced to 19-hydroxy-PGF(2alpha) with SnCl(2). 18-Hydroxy metabolites were also formed (approximately 17%). PGH(1) was metabolized to 19- and 18-hydroxy-PGH(1) in the same way. Microsomes of human seminal vesicles oxygenated arachidonate, U-44069, U-46619, U-51605, and PGH(2), similar to CYP4F8. (19R)-Hydroxy-PGE(1) and (19R)-hydroxy-PGE(2) are the main prostaglandins of human seminal fluid. We propose that they are formed by CYP4F8-catalyzed omega2-hydroxylation of PGH(1) and PGH(2) in the seminal vesicles and isomerization to (19R)-hydroxy-PGE by PGE synthase. CYP4F8 is the first described hydroxylase with specificity and catalytic competence for prostaglandin endoperoxides.


Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/metabolism , Mixed Function Oxygenases/metabolism , Prostaglandin Endoperoxides/metabolism , Seminal Vesicles/enzymology , Cytochrome P-450 Enzyme System/analysis , Humans , Male
13.
J Biol Chem ; 275(16): 11784-90, 2000 Apr 21.
Article in English | MEDLINE | ID: mdl-10766802

ABSTRACT

Cyclooxygenases catalyze the oxygenation of arachidonic acid to prostaglandin endoperoxides. Cyclooxygenase-2- and the xenobiotic-metabolizing cytochrome P450s 1A and 3A are all aberrantly expressed during colorectal carcinogenesis. To probe for a role of P450s in prostaglandin endoperoxide metabolism, we studied the 12-hydroxyheptadecatrienoate (HHT)/malondialdehyde (MDA) synthase activity of human liver microsomes and purified P450s. We found that human liver microsomes have HHT/MDA synthase activity that is concentration-dependent and inhibited by the P450 inhibitors, ketoconazole and clotrimazole with IC(50) values of 1 and 0.4 microM, respectively. This activity does not require P450 reductase. HHT/MDA synthase activity was present in purified P450s but not in heme alone or other heme proteins. The catalytic activities of various purified P450s were determined by measuring rates of MDA production from prostaglandin endoperoxide. At 50 microM substrate, the catalytic activities of purified human P450s varied from 10 +/- 1 to 0.62 +/- 0.02 min(-1), 3A4 >> 2E1 > 1A2. Oxabicycloheptane analogs of prostaglandin endoperoxide, U-44069 and U-46619, induced spectral changes in human P450 3A4 with K(s) values of 240 +/- 20 and 130 +/- 10 microM, respectively. These results suggest that co-expression of cyclooxygenase-2 and P450s in developing cancers may contribute to genomic instability due to production of the endogenous mutagen, MDA.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Fatty Acids, Unsaturated/metabolism , Malondialdehyde/metabolism , Mutagens/metabolism , Prostaglandin Endoperoxides/metabolism , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/metabolism , Animals , Catalysis , Cells, Cultured , Chromatography, Thin Layer , Heme/metabolism , Humans , Microsomes, Liver/enzymology , Prostaglandin H2 , Prostaglandins H/chemistry , Prostaglandins H/metabolism , Rabbits
14.
J Biol Chem ; 274(27): 19383-8, 1999 Jul 02.
Article in English | MEDLINE | ID: mdl-10383451

ABSTRACT

Endoperoxide antimalarials based on the ancient Chinese drug Qinghaosu (artemisinin) are currently our major hope in the fight against drug-resistant malaria. Rational drug design based on artemisinin and its analogues is slow as the mechanism of action of these antimalarials is not clear. Here we report that these drugs, at least in part, exert their effect by interfering with the plasmodial hemoglobin catabolic pathway and inhibition of heme polymerization. In an in vitro experiment we observed inhibition of digestive vacuole proteolytic activity of malarial parasite by artemisinin. These observations were further confirmed by ex vivo experiments showing accumulation of hemoglobin in the parasites treated with artemisinin, suggesting inhibition of hemoglobin degradation. We found artemisinin to be a potent inhibitor of heme polymerization activity mediated by Plasmodium yoelii lysates as well as Plasmodium falciparum histidine-rich protein II. Interaction of artemisinin with the purified malarial hemozoin in vitro resulted in the concentration-dependent breakdown of the malaria pigment. Our results presented here may explain the selective and rapid toxicity of these drugs on mature, hemozoin-containing, stages of malarial parasite. Since artemisinin and its analogues appear to have similar molecular targets as chloroquine despite having different structures, they can potentially bypass the quinoline resistance machinery of the malarial parasite, which causes sublethal accumulation of these drugs in resistant strains.


Subject(s)
Antimalarials/pharmacology , Artemisinins , Drugs, Chinese Herbal/pharmacology , Heme/metabolism , Hemoglobins/metabolism , Plasmodium falciparum/drug effects , Plasmodium yoelii/drug effects , Sesquiterpenes/pharmacology , Animals , Electrophoresis, Polyacrylamide Gel , Hemeproteins/metabolism , Male , Mice , Plasmodium falciparum/metabolism , Plasmodium yoelii/metabolism , Prostaglandin Endoperoxides/metabolism
15.
Methods Mol Biol ; 120: 257-85, 1999.
Article in English | MEDLINE | ID: mdl-10343322

ABSTRACT

The discovery of IsoPs has been an interesting development for a number of reasons, apart from the fact that it involves novel biochemistry. The simple fact that prostanoids are produced nonenzymatically in prodigous quantities in vivo and in much greater quantities than prostaglandins generated by the cyclooxygenase enzyme was a remarkable finding. The observation that detectable quantities of F2-IsoPs are present in all tissues and human biological fluids carries interesting implications. Previously, there had been little convincing evidence for the occurrence of lipid peroxidation in vivo except under unusual conditions of severe oxidative stress. However, the finding that F2-IsoPs can be easily detected in normal humans suggests a continuous level of ongoing oxidative injury that is not completely suppressed by the elaborate system of antioxidant defenses that have evolved. Another very important aspect of the discovery of IsoPs is that it has brought to the field a long sought after reliable approach to assess oxidative stress status in vivo. The continuing and expanded use of measurements of IsoPs for this purpose will contribute in a very valuable way to advancing our understanding of the role of free radicals in human disease processes. Further, the finding that these compounds are not simply markers of oxidant injury but can also exert potent biological actions both by interaction with specific receptors and, in the case of IsoLGs and cyclopentenenone IsoPs, by virtue of their chemical reactivity, has identified several new classes of molecules that are produced by free radical-induced lipid peroxidation that may mediate some of the adverse sequela of oxidant injury. The elucidation of the variety of compounds that are produced as products of the IsoP pathway and more recently the NP pathway provides vast new areas for scientific inquiry that should yield new and interesting information as this area continues to advance.


Subject(s)
Eicosanoids/metabolism , Prostaglandins/metabolism , Antioxidants/pharmacology , Docosahexaenoic Acids/metabolism , Eicosapentaenoic Acid/metabolism , Fatty Acids/metabolism , Humans , Isomerism , Lipid Peroxidation , Nervous System/metabolism , Oxidative Stress , Prostaglandin Endoperoxides
16.
Gen Pharmacol ; 32(3): 315-20, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10211585

ABSTRACT

The effects of the arachidonic acid metabolism inhibitors on the acetylcholine responses of aortae from control (CR) and deoxycorticosterone acetate (DOCA)-salt hypertensive (HR) rats were investigated. The acetylcholine decreased response observed in HR [relaxation (%): CR 95.5+/-2.7, n = 4; HR 52.0+/-6.3, n = 5, p < 0.05] was restored by the cyclooxygenase inhibitor piroxicam [relaxation (%): CR 99.8+/-0.2, n = 4; HR 86.0+/-4.0, n = 5] and by the thromboxane synthetase inhibitor and the thromboxane A2/prostaglandin H2 receptor antagonist ridogrel [relaxation (%): CR 92.1+/-4.4, n = 7; HR 93.1+/-2.0, n = 7] but not by the inhibitors of thromboxane synthetase, prostacyclin synthetase, cytochrome P-450 monooxygenase, and lipoxygenase. So, endoperoxide intermediates seem to be involved in the decreased endothelium-dependent relaxation to acetylcholine in DOCA-salt hypertension.


Subject(s)
Endothelium, Vascular/metabolism , Hypertension/metabolism , Prostaglandin Endoperoxides/metabolism , Acetylcholine/metabolism , Animals , Aorta/drug effects , Aorta/physiology , Arachidonic Acid/metabolism , Blood Pressure/drug effects , Cyclooxygenase Inhibitors/pharmacology , Desoxycorticosterone , Dose-Response Relationship, Drug , Endothelins/metabolism , Endothelium, Vascular/physiopathology , Hypertension/chemically induced , Hypertension/physiopathology , Male , Piroxicam/pharmacology , Rats , Rats, Wistar , Vasodilation
17.
Kidney Int ; 54(2): 637-45, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9690232

ABSTRACT

BACKGROUND: LGE2 is produced by the cyclooxygenase- or free radical-mediated modification of arachidonate and is formed during the oxidation of low density lipoprotein (LDL) with subsequent adduction to lysine residues in apo B. We have developed a sensitive enzyme-linked sandwich immunosorbent assay (ELISA) for detection and measurement of LGE2-protein adducts as an estimate of oxidation of plasma LDL and Lp(a). METHODS: The assay employs rabbit polyclonal antibodies directed against LGE2-protein adducts that form pyrroles, and alkaline phosphatase-conjugated polyclonal antibodies specific for apo B or apo (a). It demonstrates a high degree of specificity, sensitivity and validity. RESULTS: Epitopes characteristic for LGE2-pyrroles were quantified in patients with end-stage renal disease (ESRD) that had undergone continuous ambulatory peritoneal dialysis (CAPD) and in a gender- and age-matched control population. In addition to finding that both LDL and Lp(a) levels were elevated in CAPD patients, we also found that plasma Lp(a) but not LDL was more oxidized in CAPD patients when compared to corresponding lipoproteins from healthy subjects. Using density gradient ultra-centrifugation of plasma samples, we found that modified Lp(a) floats at the same density as total Lp(a). CONCLUSIONS: The results of this study demonstrate that oxidation of plasma Lp(a) is a characteristic of ESRD patients undergoing CAPD. This ELISA may be useful for further investigations on oxidation of lipoproteins in the circulation of specific patient populations.


Subject(s)
Kidney Failure, Chronic/metabolism , Lipoprotein(a)/metabolism , Peritoneal Dialysis, Continuous Ambulatory , Prostaglandin Endoperoxides/metabolism , Adult , Aged , Animals , Enzyme-Linked Immunosorbent Assay , Female , Humans , Lipoproteins, LDL/blood , Male , Middle Aged , Oxidation-Reduction , Rabbits
18.
Eur J Pharmacol ; 344(1): 45-8, 1998 Feb 26.
Article in English | MEDLINE | ID: mdl-9570446

ABSTRACT

The present study describes the platelet-inhibitory effects of terbogrel (5-hexenoic acid, 6-[3-[[(cyanoamino)[(1,1-dimethylethyl)amino]methylene]amino]pheny l]-6-(3-pyridinyl)-, (epsilon)-), a novel combined thromboxane A2 synthase inhibitor and thromboxane A2 receptor antagonist. Terbogrel concentration-dependently inhibited collagen (0.6 microg/ml)- and U46619 (11alpha,9alpha-epoxymethano-15(S)-hydroxy-prosta-5Z,+ ++13E-dienoic acid) (1 microM)-induced aggregation and thromboxane synthesis of washed human platelets. In this system, terbogrel exhibited an equipotent (IC50 of about 10 nM) activity as thromboxane A2 synthase inhibitor and thromboxane A2 receptor antagonist. In addition, the compound favoured prostacyclin synthesis in cultured vascular smooth muscle cells by increasing the transfer of platelet-derived prostaglandin endoperoxides. Terbogrel appears to be a compound with an equipotent molar potency as thromboxane A2 synthase inhibitor and receptor antagonist.


Subject(s)
Blood Platelets/drug effects , Enzyme Inhibitors/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Prostaglandin Endoperoxides/metabolism , Pyridines/pharmacology , Humans , Platelet Activation/drug effects , Receptors, Thromboxane/antagonists & inhibitors , Thromboxane-A Synthase/antagonists & inhibitors
19.
Article in English | MEDLINE | ID: mdl-9051715

ABSTRACT

Transcellular biosynthesis of thromboxane (Tx) A2 between vascular smooth muscle cells (SMC) and platelets has been investigated by using 14C-arachidonic acid (AA) radiolabeled rat SMC (or platelets) and the fate of the label in phospholipids and eicosanoid fractions was studied using radioimmunoassay (RIA) and thin-layer chromatography (TLC). Stimulation of SMC with interleukin-1 beta (IL-1 beta) resulted in production of cyclooxygenase metabolites (e.g. 6-keto-PGF1 alpha, PGE2, PGF2 alpha, PGD2), 15-, 11-, 5-HETE, and free AA1 with a coincident decline of phosphatidylcholine (PC) in SMC. IL-1 beta did not induce TXB2 production, a stable metabolite of TXA2 measured by TLC and radioimmunoassay, either in human platelets from 0.01-100 U/ml for 1 h or in SMC for 24 h. However, human platelets converted exogenous PGH2 to TXA2 despite cyclooxygenase inhibition or PGH2 receptor blockade. Furthermore, TXB2 was produced in large quantities during co-incubation of IL-1 beta-stimulated SMC with human platelets for 30 min in concert with a significant decrease of 6-keto-PGF1 alpha and eicosanoids (PGE2, PGF2 alpha and PGD2) compared with control (P < 0.01). Pretreatment of SMC with cycloheximide and actinomycin not only inhibited IL-1 beta-induced eicosanoid synthesis and phospholipid breakdown but also diminished TXB2 production when co-incubated with platelets. These data suggest that a cell-cell interaction, i.e. platelet utilizing SMC-derived endoperoxides for its TXA2 production, might cause an excess thromboxane A2 synthesis.


Subject(s)
Blood Platelets/metabolism , Cell Communication , Interleukin-1/pharmacology , Muscle, Smooth, Vascular/metabolism , Thromboxane A2/biosynthesis , 6-Ketoprostaglandin F1 alpha/metabolism , Animals , Arachidonic Acid/metabolism , Aspirin/pharmacology , Blood Platelets/drug effects , Cells, Cultured , Coculture Techniques , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Humans , Muscle, Smooth, Vascular/cytology , Phospholipids/metabolism , Prostaglandin Endoperoxides/metabolism , Prostaglandin H2 , Prostaglandins H/metabolism , Rats , Recombinant Proteins/pharmacology , Thrombin/pharmacology
20.
Eur J Pharmacol ; 317(1): 91-6, 1996 Dec 12.
Article in English | MEDLINE | ID: mdl-8982724

ABSTRACT

Thromboxane A2/prostaglandin endoperoxide receptor antagonists were studied in rat and guinea-pig aortas contracted with U-46619 (9,11-dideoxy-11 alpha,9 alpha-epoxymethanoprostaglandin F2 alpha) or 8-epi-prostaglandin F2 alpha. In rat aorta, the antagonists competitively inhibited contractions evoked by either agonist with a rank order of potency as follows: BMS-180291 ([1s-(exo,exo)]-2-[[3-[4-[(pentylamino) carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]-benz enepropanoic acid) > or = SQ 29,548 ([1s-[1 alpha,2 beta-(5z), 3 beta,4 alpha)]-7-[3-[[2-[(phenylamino)carbonyl]hydrozino] methyl]-7-oxobicyclo-[2.2.1]hept-2-yl]-5-heptanoic acid) > daltroban (4-[2-(4-chlorobenzenesulfonylamino) methyl]-benzene acetic acid) > or = SQ 30,741 ([1s-[1 beta,2 alpha(5z),3 alpha,4 beta]]-7-[3-[[[[(oxa)amino]acetyl] amino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl-5-heptanoic acid) = AA-2414 (2,4,5-trimethyl-3,6-dioxo-zeta-phenyl-1,4-cyclohexadien-1-heptano ic acid). In guinea-pig aorta, the antagonists competitively antagonized contractions elicited by either agonist with the following rank order of potency: SQ 29,548 = AA-2414 > or = SQ 30,741 > daltroban. Antagonism by BMS-180291 in guinea-pig aorta was not strictly competitive. These findings indicate that thromboxane A2/prostaglandin endoperoxide receptors in rat aortas are different from those in guinea pigs. Because the actions of both agonists were equivalently antagonized by each of the antagonists in both rat and guinea-pig aortas, the results do not support the hypothesis that U-46619 and 8-epi-prostaglandin F2 alpha elicit contractions via different receptor subtypes in the aorta.


Subject(s)
Aorta, Thoracic/metabolism , Prostaglandin Antagonists/pharmacology , Prostaglandin Endoperoxides/metabolism , Receptors, Prostaglandin/agonists , Receptors, Prostaglandin/antagonists & inhibitors , Receptors, Thromboxane/agonists , Receptors, Thromboxane/antagonists & inhibitors , Thromboxane A2/metabolism , Animals , Aorta, Thoracic/drug effects , Guinea Pigs , In Vitro Techniques , Male , Rats , Rats, Sprague-Dawley , Receptors, Prostaglandin/metabolism , Receptors, Thromboxane/metabolism
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