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At least 19 recordsLinked to original sources
PubMed1997

Aspirin induces increased expression of both prostaglandin H synthase-1 and prostaglandin H synthase-2 in cultured human placental trophoblast.

OBJECTIVE: We tested the hypothesis that aspirin affects trophoblast like other epithelial cells do, by inhibiting prostanoid production, inducing prostaglandin H synthase-2 expression, and enhancing secretion of 15-hydroxyeicosatetraenoic acid. STUDY DESIGN: Cytotrophoblast from placentas (n = 15) of uncomplicated singleton pregnancies were cultured in medium 199 for 4 to 72 hours in the presence or absence of aspirin. RESULTS: Aspirin (10(-4) M) inhibited (p < 0.01) average trophoblast prostaglandin E2 release by 60% and thromboxane B2 by 86%. Western immunoblotting showed the prostaglandin H synthase-1 was constitutively expressed in cytotrophoblast, and aspirin treatment caused a twofold increase in prostaglandin H synthase-1 expression. Prostaglandin H synthase-2 was also constitutively expressed in untreated cytotrophoblast but at lower levels than prostaglandin H synthase-1. Aspirin enhanced prostaglandin H synthase-2 expression in trophoblast cultures, but prostaglandin H synthase-2 contributed a range of only 10% to 33% (n = 4) of the total cellular prostaglandin H synthase protein pool even after aspirin induction. The increased prostaglandin H synthase expression depended on both transcription and translation because actinomycin D and cycloheximide each inhibited the increased prostaglandin H synthase protein expression after aspirin treatment. The aspirin induction of prostaglandin H synthase was accompanied by decreased release of 15-hydroxyeicosatetraenoic acid. CONCLUSIONS: Trophoblast differs from other cells studied because aspirin enhances expression of both prostaglandin H synthase-1 and prostaglandin H synthase-2 isozymes while decreasing, instead of increasing, the secretion of 15-hydroxyeicosatetraenoic acid. The aspirin effects on prostaglandin H synthase synthesis and 15-hydroxyeicosatetraenoic acid release in trophoblast suggest that the mechanisms of action for aspirin in the prophylaxis of preeclampsia may be more diverse than simply altering platelet thromboxane production.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2001

Oxidation of prostaglandin H(2) and prostaglandin H(2) analogues by human cytochromes P450: analysis of omega-side chain hydroxy metabolites and four steroisomers of 5-hydroxyprostaglandin I(1) by mass spectrometry.

The objective was to examine the NADPH-dependent oxygenation of prostaglandin H(2) (PGH(2)) and three PGH(2) analogues, 9,11-diazo-15-deoxy-PGH(2) (U51605), 9,11-epoxymethano-PGH(2) (U44069), and 11,9-epoxymethano-PGH(2) (U46619), by cytochromes P450, and to characterize the metabolites by mass spectrometry. CYP2C19, CYP4A11, CYP4F8, and liver and renal cortical microsomes oxidized the omega-side chain of U44069, U46619, and U51605, whereas only CYP4F8 oxidized the omega-side chain of PGH(2). PGH(2) was transformed to four stereoisomers of 5-hydroxy-PGI(1) by recombinant cytochromes P450. CYP4F8 formed the 5-hydroxy-PGI(1) isomers in small amounts compared to the 19-hydroxy metabolites of PGH(2). Isomers of 5-hydroxy-PGI(1) and 6-keto-PGF(1 alpha) were detectable when PGH(2) decomposed in the presence of hemin, hemoglobin, or heat-inactivated microsomes. 5-Hydroxy-PGI(1) is likely formed from PGH(2) in a pseudo-enzymatic reaction involving homolytic scission of the endoperoxide and formation of an ether between C-9 and C-6 and a carbon-centered radical at C-5, which reacts with molecular oxygen. CYP4F8 catalyzes 19-hydroxylation of PGH(2), but the absolute configuration of the 19-hydroxy group is unknown, whereas human seminal fluid contains (19R)-hydroxy-PGE(2). CYP4F8 was found to metabolize U51605 to 90% of the (19R)-hydroxy metabolite, providing further evidence in favor of a role of CYP4F8 in biosynthesis of (19R)-hydroxy PGE in human seminal vesicles. We conclude that omega-side chain hydroxylation of PGH(2) analogues may be catalyzed by many different cytochromes P450, but only CYP4F8 oxidizes the omega-side chain of PGH(2) efficiently.

Animals↗
PubMed1998

Structural characterization of arachidonyl radicals formed by prostaglandin H synthase-2 and prostaglandin H synthase-1 reconstituted with mangano protoporphyrin IX.

A tyrosyl radical generated in the peroxidase cycle of prostaglandin H synthase-1 (PGHS-1) can serve as the initial oxidant for arachidonic acid (AA) in the cyclooxygenase reaction. Peroxides also induce radical formation in prostaglandin H synthase-2 (PGHS-2) and in PGHS-1 reconstituted with mangano protoporphyrin IX (MnPGHS-1), but the EPR spectra of these radicals are distinct from the initial tyrosyl radical in PGHS-1. We have examined the ability of the radicals in PGHS-2 and MnPGHS-1 to oxidize AA, using single-turnover EPR studies. One wide singlet tyrosyl radical with an overall EPR line width of 29-31 gauss (G) was generated by reaction of PGHS-2 with ethyl hydroperoxide. Anaerobic addition of AA to PGHS-2 immediately after formation of this radical led to its disappearance and emergence of an AA radical (AA.) with a 7-line EPR, substantiated by experiments using octadeuterated AA. Subsequent addition of oxygen resulted in regeneration of the tyrosyl radical. In contrast, the peroxide-generated radical (a 21G narrow singlet) in a Y371F PGHS-2 mutant lacking cyclooxygenase activity failed to react with AA. The peroxide-generated radical in MnPGHS-1 exhibited a line width of 36-38G, but was also able to convert AA to an AA. with an EPR spectrum similar to that found with PGHS-2. These results indicate that the peroxide-generated radicals in PGHS-2 and MnPGHS-1 can each serve as immediate oxidants of AA to form the same carbon-centered fatty acid radical that subsequently reacts with oxygen to form a hydroperoxide. The EPR data for the AA-derived radical formed by PGHS-2 and MnPGHS-1 could be accounted for by a planar pentadienyl radical with two strongly interacting beta-protons at C10 of AA. These results support a functional role for peroxide-generated radicals in cyclooxygenase catalysis by both PGHS isoforms and provide important structural characterization of the carbon-centered AA..

Arachidonic Acid↗
PubMed2001

Prostaglandin H synthase-2 inhibitors interfere with prostaglandin H synthase-1 inhibition by nonsteroidal anti-inflammatory drugs.

Ram seminal vesicle microsomes, a rich source of prostaglandin H synthase-1, were incubated with 100 nM of the prostaglandin H synthase-2 inhibitors N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398) and 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl) thiophene (DuP-697) prior to exposure to the prostaglandin H synthase inhibitors aspirin, indomethacin, ibuprofen or naproxen. Activity of the enzyme was measured by following the conversion of arachidonic acid to prostaglandin E(2) and prostaglandin F2alpha. Although prostaglandin H synthase-1 activity was not altered by these concentrations of the prostaglandin H synthase-2 inhibitors, it was found that exposure to these agents prior to aspirin or indomethacin (irreversible prostaglandin H synthase inhibitors) significantly attenuated the inhibition obtained by the latter inhibitors. On the other hand, the same concentrations of the prostaglandin H synthase-2 inhibitors did not interfere with prostaglandin H synthase-1 inhibition that was induced by naproxen or ibuprofen (competitive prostaglandin H synthase inhibitors). Attenuation of the indomethacin inhibition of prostaglandin H synthase-1 by prostaglandin H synthase-2 inhibitors was observed only when the microsomes were pre-exposed to DuP-697 or NS-398 in the absence, but not in the presence, of arachidonic acid. The effect of DuP-697 was found to be irreversible, however, washing away the agent reversed the action of NS-398. Similar phenomena have been reported by us in bovine aortic endothelial cells and in human dermal fibroblasts. Attenuation of the inhibition by aspirin and indomethacin, without altering the enzyme's basal activity or the inhibition induced by ibuprofen or naproxen may suggest the possibility that the prostaglandin H synthase-2 specific inhibitors DuP-697 and NS-398 affect prostaglandin H synthase-1 by interaction with a site different from the enzyme's catalytic site.

Animals↗
PubMed1998

The dynamics of prostaglandin H synthases. Studies with prostaglandin h synthase 2 Y355F unmask mechanisms of time-dependent inhibition and allosteric activation.

Prostaglandin H synthases (PGHSs) catalyze the conversion of arachidonic acid to prostaglandins. In this report, we describe the effect of a PGHS2 Y355F mutation on the dynamics of PGHS2 catalysis and inhibition. Tyr355 is part of a hydrogen-bonding network located at the entrance to the cyclooxygenase active site. The Y355F mutant exhibited allosteric activation kinetics in the presence of arachidonic acid that was defined by a curved Eadie-Scatchard plot and a Hill coefficient of 1.36 +/- 0.05. Arachidonic acid-induced allosteric activation has not been directly observed with wild type PGHS2. The mutation also decreased the observed time-dependent inhibition by indomethacin, flurbiprofen, RS-57067, and SC-57666. Detailed kinetic analysis showed that the Y355F mutation decreased the transition state energy associated with slow-binding inhibition (EIdouble dagger) relative to the energy associated with catalysis (ESdouble dagger) by 1.33, 0.67, and 1.06 kcal/mol, respectively, for indomethacin, flurbiprofen, and RS-57067. These observations show Tyr355 to be involved in the molecular mechanism of time-dependent inhibition. We interpret these results to indicate that slow binding inhibitors and the Y355F mutant slow the rate and unmask intrinsic, dynamic events associated with product formation. We hypothesize that the dynamic events are the equilibrium between relaxed and tightened organizations of the hydrogen-bonding network at the entrance to the cyclooxygenase active site. It is these rearrangements that control the rate of substrate binding and ultimately the rate of prostaglandin formation.

Allosteric Regulation↗
PubMed1993

Serum and glucocorticoid regulation of gene transcription and expression of the prostaglandin H synthase-1 and prostaglandin H synthase-2 isozymes.

Mitogenic stimulation has been shown to increase both prostaglandin H (PGH) synthase-1 (PGHS-1) and PGH synthase-2 (PGHS-2) mRNA levels, although the time course and magnitude of induction are different for the two genes. To investigate the mechanism for mRNA induction, we conducted nuclear run-off assays of these two genes in 3T3 cells and correlated mitogen-induced changes in PGHS gene transcription with changes in PGHS mRNA and PGHS isozyme expression. We also examined the mechanism for glucocorticoid inhibition of PGHS mRNA expression and the effects of glucocorticoids on PGHS isozyme expression. Serum stimulation of quiescent 3T3 cells led to a sequential increase in PGHS-2 gene transcription, PGHS-2 mRNA, and PGHS-2 enzyme levels. PGHS-2 gene transcription increased over 25-fold within 30 min of serum addition resulting in an over 70-fold increase in PGHS-2 mRNA by 1 h, and maximal PGHS-2 enzyme expression by 2 h. Increased PGHS-2 isozyme expression thus appears to depend on transcriptional activation of the gene. Transcription of the PGHS-2 gene declined after 30 min, and PGHS-2 mRNA levels declined similarly after 1 h, leading to a return of PGHS-2 levels to near basal levels by 6 h. Glucocorticoids, which previously have been shown to inhibit mitogen-stimulated increases in PGHS-2 levels, were found to inhibit serum-stimulated increases in PGHS-2 gene transcription by 70%, resulting in a 70% reduction in peak serum-stimulated PGHS-2 mRNA levels also. Western blotting with PGHS-2 specific antisera demonstrated that while dexamethasone simply reduced PGHS-2 mRNA levels, it completely suppressed expression of PGHS-2 protein. The coincidental reduction in PGHS-2 transcription, PGHS-2 mRNA, and enzyme levels by dexamethasone, provides further support for the hypothesis that control of transcription is one primary control mechanism for regulating PGHS-2 expression. That complete suppression of PGHS-2 enzyme expression occurs following partial suppression of PGHS-2 mRNA, however, suggests that other mechanisms may also contribute to the glucocorticoid effect. A small, but reproducible, increase in transcription of the PGHS-1 gene occurred 3 h following serum stimulation, coincident with a three- to fourfold increase in PGHS-1 mRNA; PGHS-1 mRNA remained elevated for at least 3 h. Dexamethasone reduced, but did not completely inhibit, the serum-stimulated increases in PGHS-1. However, changes in PGHS-1 mRNA were not accompanied by detectable changes in PGHS-1 protein in the presence or absence of dexamethasone.

3T3 Cells↗
PubMed1987

Immunochemical and kinetic evidence for two different prostaglandin H-prostaglandin E isomerases in sheep vesicular gland microsomes.

Splenic lymphocytes from mice immunized with a partially purified prostaglandin (PG) H-PGE isomerase from sheep vesicular glands were fused with SP2/0-Ag14 myeloma cells. Two spleen cell-myeloma hybrids (hei-7 and hei-26) were selected and cloned. The mouse antibodies secreted by the two hybrids, IgG1 (hei-7) and IgG1 (hei-26), caused immunoprecipitation of a maximum of 45 and 22%, respectively, of the solubilized PGH-PGE isomerase activity of sheep vesicular gland; immunoprecipitation of activity by the two antibodies was additive. The antigens reactive with IgG1 (hei-7) and IgG1 (hei-26) were identified as proteins with Mr = 17,500 and 180,000, respectively, by Western transfer blotting or sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitated 125I-labeled microsomes. The PGH-PGE isomerase activities precipitated by IgG1 (hei-7) and IgG1 (hei-26) exhibited different kinetic properties with respect to time course, Km for PGH2, and concentration dependence for GSH. No significant GSH-S-transferase activity was present in these immunoprecipitates. These data indicate that there are at least two different proteins in sheep vesicular gland microsomes capable of catalyzing GSH-dependent PGH-PGE isomerase reactions. IgG1 (hei-7), but not IgG1 (hei-26), caused coprecipitation of PGH synthase and PGH-PGE isomerase activities when incubated with intact right-side-out vesicular gland microsomes. Thus, the epitope for IgG1 (hei-7) is located on the cytoplasmic surface of those microsomal spheres which contain PGH synthase. This latter finding suggests that the isomerase reactive with IgG1 (hei-7) is involved in PGE synthesis in sheep vesicular glands.

Animals↗
PubMed1991

Studies on the reduction of endogenously generated prostaglandin G2 by prostaglandin H synthase.

Prostaglandin H synthase oxidizes arachidonic acid to prostaglandin G2 (PGG2) via its cyclooxygenase activity and reduces PGG2 to prostaglandin H2 by its peroxidase activity. The purpose of this study was to determine if endogenously generated PGG2 is the preferred substrate for the peroxidase compared with exogenous PGG2. Arachidonic acid and varying concentrations of exogenous PGG2 were incubated with ram seminal vesicle microsomes or purified prostaglandin H synthase in the presence of the reducing cosubstrate, aminopyrine. The formation of the aminopyrine cation free radical (AP.+) served as an index of peroxide reduction. The simultaneous addition of PGG2 with arachidonic acid did not alter cyclooxygenase activity of ram seminal vesicle microsomes or the formation of the AP.+. This suggests that the formation of AP.+, catalyzed by the peroxidase, was supported by endogenous endoperoxide formed from arachidonic acid oxidation rather than by the reduction of exogenous PGG2. In addition to the AP.+ assay, the reduction of exogenous versus endogenous PGG2 was studied by using [5,6,8,9,11,12,14,15-2H]arachidonic acid and unlabeled PGG2 as substrates, with gas chromatography-mass spectrometry techniques to measure the amount of reduction of endogenous versus exogenous PGG2. Two distinct results were observed. With ram seminal vesicle microsomes, little reduction of exogenous PGG2 was observed even under conditions in which all of the endogenous PGG2 was reduced. In contrast, studies with purified prostaglandin H synthase showed complete reduction of both exogenous and endogenous PGG2 using similar experimental conditions. Our findings indicate that PGG2 formed by the oxidation of arachidonic acid by prostaglandin H synthase in microsomal membranes is reduced preferentially by prostaglandin H synthase.

Animals↗
PubMed2005

Localization of prostaglandin H synthase, prostaglandin dehydrogenase, corticotropin releasing hormone and glucocorticoid receptor in rhesus monkey fetal membranes with labor and in the presence of infection.

Prostaglandins (PGs) play a central role in primate parturition by their actions on uterine contractility and on cervical ripening. Rhesus monkey placentation is hemochorial and the endocrine events surrounding parturition are qualitatively similar to human pregnancy. Although there is an increase in PG production before the onset of labor, little is known about the cellular localization of the PGH synthase (PGHS) or the 15-hydroxy PG dehydrogenase (PGDH) in the fetal membranes of nonhuman primates and whether it changes at term in spontaneous labor or during preterm labor associated with infection. Placental corticotropin releasing hormone (CRH) and the glucocorticoid receptor (GR) have also been implicated as mediators in parturition by virtue of their roles in PG production. We utilized immunohistochemical methods to localize the inducible isoform PGHS-2, PGDH, GR and CRH in rhesus monkey amnion, chorion and attached decidua. Tissues were obtained at cesarean section during late pregnancy, in spontaneous labor at term and in premature labor induced by Group B streptococcal intraamniotic infection. Specific staining for immunoreactive (ir)-PGHS-2 was observed in amnion epithelial and mesenchymal cells and to a lesser extent in chorion and decidua. In contrast, ir-PGDH was localized primarily to the extravillous trophoblast layer of chorion. GR was localized to both the cytoplasm and nucleus of amnion epithelial cells, subepithelial fibroblasts, chorion trophoblasts and in decidua. Immunostaining for CRH was found in amnion and in scattered decidual cells but was most intense in the chorion trophoblast layer. There was no demonstrable change in this overall pattern of immunostaining in association with the onset of labor at term except for a decrease in staining for ir-PGDH in chorion. Experimental Group B streptococcal chorioamnionitis resulted in preterm labor and extensive necrosis of extravillous trophoblast cells with subsequent loss of chorionic ir-PGDH and relative sparing of ir-PGHS-2 in amnion epithelium which favors the net production of PGs. The expression pattern of these effectors in the rhesus monkey fetal membranes points to a functional role of PGs and glucocorticoids in the process of term and preterm parturition which is similar to that in human pregnancy.

Animals↗
PubMed1996

Peroxidative oxidation of leuco-dichlorofluorescein by prostaglandin H synthase in prostaglandin biosynthesis from polyunsaturated fatty acids.

Prostaglandin H synthase can oxidize arachidonic acid with leuco-dichlorofluorescein as reducing cosubstrate. Addition of 0.5 mM phenol increases the oxidation of leuco-dichlorofluorescein to dichlorofluorescein 5-fold, probably by acting as a cyclic intermediate in the oxidation. Tetramethyl-p-phenylenediamine is also oxidized as cosubstrate. Its oxidation is not influenced by phenol. A stoichiometry of close to one mole of tetramethyl-p-phenylenediamine or leuco-dichlorofluorescein consumed per mole of arachidonic acid was found in the initial phase of the reaction. In the presence of phenol + leuco-dichlorofluorescein, the oxidation rate of arachidonic acid is about 40% lower than with phenol alone as cosubstrate. Since dichlorofluorescein has a molar extinction coefficient of 91 . 10(3) at 502 nm, the oxidation of less than 1 microM leuco-dichlorofluorescein can be detected spectrophotometrically. The rate of extinction change with leuco-dichlorofluorescein (at 502 nm) is about 4-fold more rapid than with tetramethyl-p-phenylenediamine (at 611 nm). With this spectrophotometric assay we have confirmed that arachidonic acid, linolenic acid, adrenic acid, gamma-linolenic acid, eicosapentaenoic acid, are substrates for prostaglandin H synthase with decreasing reaction rates in the mentioned order. The same order of reaction rates were found when oxygen consumption was measured. The assay also shows that docosahexaenoic acid is substrate for the enzyme. The reaction rate of the enzyme evidently is decreased both by a n-3 double bond and by deviation from a 20 carbon chain length of the fatty acid substrate.

Animals↗
PubMed1989

Activation of aromatic amines by prostaglandin H synthase.

Prostaglandin H synthase catalyzes the first step in the synthesis of prostaglandins from arachidonic acid. The peroxidase activity of this enzyme can support the oxidation of xenobiotics, particularly aromatic amines. This pathway of metabolism may contribute to the activation of carcinogenic aromatic amines in target tissues such as the skin, lung, and bladder. In this review, recent work on this subject is summarized. I emphasize the elucidation of the structures of aromatic amine oxidation products, and their interactions with biological macromolecules. Prostaglandin H synthase supports the activation of benzidine to a mutagenic species in the Ames (Salmonella typhimurium) test, and our studies of the mechanism of this activation are described.

Amines↗
PubMed2002

On the interaction of specific prostaglandin H synthase-2 inhibitors with prostaglandin H synthase-1.

Previous studies with both intact cells and ram seminal vesicles microsomes have shown that the specific PGHS-2 inhibitors NS-398 (N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide) and DuP-697 (5-bromo-2[4-fluorophenyl]-3-[4-methylsulfonylphenyl]-thiophene) attenuate the inhibition of PGHS-1 caused by aspirin and indomethacin. This effect occurs at concentrations of PGHS-2 inhibitors that do not inhibit the cyclooxygenase activity of PGHS-1. Here we study the effect of NS-398 and ibuprofen, a nonspecific inhibitor, on the indomethacin-induced inhibition of purified PGHS-1 and compare this effect with that observed with microsomal enzyme. Dissociation constants are obtained for the interaction of NS-398 with the purified and microsomal PGHS-1 using curve fitting of experimental data on the interaction of indomethacin with the enzyme. The dissociation constants for ibuprofen and NS-398 for interaction with PGHS-1 are similar. This finding indicates that specific PGHS-2 inhibitors are similar to ibuprofen in their ability to compete with aspirin, an irreversible time-dependent inhibitor of PGHS-1 often used for prevention of spontaneous thrombosis. Importantly, the concentrations at which PGHS-2 inhibitors attenuate the inhibition induced by aspirin and indomethacin are well below those required to cause inhibition of PGHS-1. Our results suggest that arachidonic acid not only competes with PGHS-2 inhibitors for binding to the cyclooxygenase site of PGHS-1 but it also reduces the affinities of PGHS-1 for these inhibitors by an additional, as yet unresolved mechanism.

Animals↗
PubMed1985

Formation of unique arylamine:DNA adducts from 2-aminofluorene activated by prostaglandin H synthase.

Prostaglandin H synthase in the presence of arachidonic acid catalyzes the peroxidative metabolism of 2-aminofluorene (2-AF) to an electrophile(s) which binds covalently to calf thymus DNA in vitro. Moreover, this electrophile(s) appears distinct from the classical 2-AF-derived electrophiles, N-hydroxy-2-AF and the 2-AF nitrenium ion. Both the prostaglandin H synthase:arachidonic acid and horseradish peroxidase:hydrogen peroxide systems were used to investigate the binding of [3H]-2-AF to DNA and the nature of the DNA adducts formed from peroxidative activation of 2-AF. Modification of DNA by N-hydroxy-2-AF under mildly acidic conditions was used as a reference system in these studies and yielded a single 2-AF:nucleoside adduct, identified as N-(deoxyguanosin-8-yl)-2-AF (C8-dGuo-AF). Enzymatic hydrolysis of DNA modified by 2-AF activated in either of the peroxidase systems liberated 2-AF:nucleoside adducts that differed considerably from C8-dGuo-AF in chromatographic and extraction properties. C8-dGuo-AF from DNA hydrolysates was easily extracted into n-butyl alcohol and adsorbed by Sephadex LH-20 columns. In contrast, the peroxidase-derived adducts were poorly extracted into n-butyl alcohol and were not retained on Sephadex LH-20 columns. Experimental evidence suggests the peroxidase-derived adducts may possess a negative charge at neutral pH. Since C8-dGuo-AF is the only 2-AF:nucleoside adduct formed when 2-AF is activated via N-hydroxylation, these new adducts represent a marker unique to peroxidative activation of 2-AF. Therefore, 2-AF:DNA adducts can be used as a differential end point with which to assess the relative roles of N-hydroxylation and peroxidation in the metabolic activation of 2-AF in cell culture and in target tissues in vivo.

Animals↗
PubMed1994

Effect of cholera toxin and pertussis toxin on prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteinase production by human monocytes.

Activation of human monocytes induces the production of matrix metalloproteinases (MMPs) through a prostaglandin E2 (PGE2)-cAMP-dependent pathway. Since G-proteins have been documented to modulate adenylyl cyclase, we examined the effect of G-protein ADP-ribosylating agents, cholera toxin (CT) and pertussis toxin (PT), on the signal transduction pathway that culminates in the production of monocyte MMPs. Although CT elevated cAMP levels in both unstimulated and concanavalin A (Con A)-stimulated monocytes, it enhanced the production of prostaglandin H synthase-2 (PGH synthase-2, PGHS-2) protein, prostaglandins, interstitial collagenase, and 92-kDa type IV collagenase/gelatinase only in Con A-stimulated monocytes. Additionally, the indomethacin-mediated suppression of Con A-induced monocyte interstitial collagenase and 92-kDa type IV collagenase/gelatinase production could be reversed by CT. In contrast to the actions of CT, PT treatment suppressed the levels of cAMP, PGHS-2, PGE2, interstitial and 92-kDa type IV collagenase/gelatinase in Con A-stimulated monocytes. The regulation of MMP production by these toxins appears to be mediated primarily through their effect on adenylyl cyclase since the release of arachidonic acid was relatively unaffected by these agents. These findings provide evidence that G-proteins may be involved in either the enhancement or suppression of the eicosanoid-cAMP-dependent signal transduction pathway that results in the production of monocyte MMPs.

Adenylate Cyclase Toxin↗
PubMed1997

Secretory leukocyte protease inhibitor suppresses the production of monocyte prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteinases.

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor found in fluids lining mucosal surfaces. In addition to its primary function as an antiprotease, SLPI may also influence cellular functions associated with enzyme synthesis and retroviral infection. In this study, SLPI was examined for its effect on signaling events involved in the production of matrix metalloproteinases (MMPs) by monocytes. Addition of SLPI before stimulation with concanavalin A or LPS resulted in a significant inhibition of monocyte prostaglandin H synthase-2 (PGHS-2), a pivotal enzyme in the PGE2-cAMP dependent pathway of monocyte MMP synthesis. Suppression of PGHS-2 was detected with 0.1 microg/ml of SLPI with a substantial inhibition at 1 and 10 micro/ml. Attenuation of PGHS-2 by SLPI was accompanied by decreased production of PGE2 resulting in the suppression of interstitial collagenase (MMP-1) and gelatinase B (MMP-9) that was reversed by PGE2 or Bt2cAMP. The inhibitory effect of SLPI was largely independent of its antiprotease activity because SLPI muteins, with significantly lower antiprotease activity, also suppressed the induction of PGHS-2 and MMPs. The inhibitory effects of SLPI did not involve the modulation of monokine production since TNF-alpha and IL-10 were unaffected. These findings demonstrate that SLPI also functions as a potent antiinflammatory agent by interfering with the signal transduction pathway leading to monocyte MMP production.

Blotting, Northern↗
PubMed1998

Paradoxical effects of resveratrol on the two prostaglandin H synthases.

Prostaglandin H synthase (PGHS) is the primary enzyme responsible for the biosynthesis of prostaglandins and thromboxanes. Of the two isoenzymes of PGHS, PGHS-1 is constitutively expressed and PGHS-2 is inducible by mitogens or other inflammatory stimuli. Constitutive expression of PGHS-2 in neoplastic tissues has been implicated in carcinogenesis. Resveratrol, a lignan, was recently shown to be an anticarcinogen that selectively inhibits PGHS-1. In vitro experiments to resolve these seemingly paradoxical observations revealed that resveratrol is not only an inhibitor of PGHS-1 but also is an activator of PGHS-2. Resveratrol non-competitively inhibited PGHS-1 with a K1 of 26 +/- 2 microM but enhanced the PGHS-2 activity nearly twofold. Additionally, resveratrol did not serve as a reducing co-substrate for the peroxidase activities of either enzyme despite being an easily oxidizable phenolic compound. Resveratrol inhibited the peroxidase activity of PGHS-1 (IC50 = 15 microM) better than that of PGHS-2 (IC50 = > 200 microM). Inhibition of the perxidase activity but not the cyclooxygenase activity of PGHS-2 resulted in the production of PGG2 from arachidonic acid. A plausible relationship between these observation and the anticarcinogenic activity of resveratrol is discussed.

Animals↗
PubMed1997

Delayed release of prostaglandins from arachidonic acid and kinetic changes in prostaglandin H synthase activity on the induction of prostaglandin H synthase-2 after lipopolysaccharide-treatment of RAW264.7 macrophage-like cells.

In a mouse macrophage-like cell line, RAW264.7, arachidonic acid was cleaved within 30 min of lipopolysaccharide (LPS)-treatment. However, prostaglandin (PG) synthesis did not accompany this cleavage, being delayed by 4 h, although significant PGH synthase (PGHS) activity was detected in the lysate of LPS-nontreated cells. The K(m) value of this basal PGHS activity toward arachidonic acid was more than one hundred-fold higher than that for the lysate of cells treated with LPS for 4 h. Changes in the sensitivity of the PGHS activity to nonsteroidal antinflammatory drugs after LPS-treatment also suggested induction of PGHS with different properties from that in the case of basal PGHS. The concomitant increase in PGH synthase (PGHS) activity with the induction of PGHS-2 protein after LPS-treatment suggested a contribution by PGHS-2 to the delayed synthesis of PGs in LPS-treated macrophage cells. As for PGHS in the control cells without LPS-treatment, a different K(m) value from that of PGHS-1 and the lack of cross-reactivity to anti-PGHS-1 antibodies suggested that this basal PGHS was different from the typical PGHS-1. The lower affinity of this enzyme for arachidonic acid might be the reason for the failure to release PGs by the cells without LPS-treatment.

Animals↗
PubMed2002

Synthesis of isotopically labeled arachidonic acids to probe the reaction mechanism of prostaglandin H synthase.

Prostaglandin H synthase (PGHS) catalyzes the conversion of arachidonic acid to prostaglandin G(2) in the cyclooxygenase reaction. The first step of the mechanism has been proposed to involve abstraction of the pro-S hydrogen atom from C13 to generate a pentadienyl radical spanning C11-C15. We report here the synthesis of six site-specifically deuterated arachidonic acids to investigate the structure of the radical intermediate. The preparation of these compounds was achieved using a divergent scheme that involved one advanced intermediate for all targets. The synthetic design introduced the label late in the routes and allowed the utilization of common synthetic intermediates in the preparation of various targets. Both 13(R)- and 13(S)-deuterium-labeled arachidonic acids were synthesized in high enantiomeric purity as deduced from soybean lipoxygenase assays and mass spectrometric analysis of the resulting enzymatic products. Each synthetic compound was reacted under anaerobic conditions with the wide singlet tyrosyl radical of PGHS-2 to generate a radical intermediate that was analyzed by EPR. Deuterium substitution at positions 11, 13(S), and 15 resulted in the loss of one hyperfine interaction, indicating that the protons at these positions interact with the unpaired electron. Simulation of the spectra was achieved with one set of parameters that are consistent with the assignment of a pentadienyl radical. Use of 16-[(2)H(2)]-arachidonic acid indicated that only one of the protons at C16 gives rise to a strong hyperfine interaction. The findings are discussed in the context of two proposed mechanisms for the cyclooxygenase reaction.

Arachidonic Acids↗