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.     

Inhibition of prostaglandin synthesis by lysolecithin

Exogenous lysolecithin inhibits prostaglandin E₂ synthesis from arachidonic acid in bovine seminal vesicle microsomes at plausible physiological levels (lysolecithin-to-protein ratios ? 0.03 [w/w]) by inhibiting fatty acid cyclo-oxygenase activity. Structurally defined lysolecithins with varying fatty acid chain length exhibit varying effectiveness as inhibitors. Addition of equimolar quantities of free fatty acid lowers the lysolecithin concetration required for inhibition. Exogenous lysolecithin inhibits unstimulated and thrombin-stimulated prostaglandin E₂ synthesis from endogenous substrate in SVBalb/3T3 cells. Serum treatment of SVBalb/3T3 cells, which generates endogenous lysolecithin and free fatty acids, decreases the efficiency of conversion of free arachidonic acid to prostaglandins. These results suggest a possible role for the products of phospholipase A2 action in the regulation of prostaglandin synthesis.     

Studies on the covalent binding of an intermediate(s) in prostaglandin biosynthesis to tissue macromolecules.

We investigated the covalent binding of intermediates in prostaglandin biosynthesis to tissue macromolecules. Following incubation of [1-14C]arachidonic acid with the microsomal fraction from guinea pig lung, ram or bovine seminal vesicle, human platelets, rabbit kidney, or rat stomach fundus, the amount of covalent binding of arachidonic acid metabolites expressed as percentage of total arachidonic acid metabolized varied from tissue to tissue ranging from 3% in human platelets to 18.2% in ram seminal vesicles. In general, the thromboxane synthesizing tissues had less covalently bound metabolites than the other tissues. The amount of covalently bound metabolites was increased in the guinea pig lung microsomes when the thromboxane synthetase inhibitor, N-0164, was added to the incubation mixture. The covalent binding of arachidonic acid metabolite(s) was greatly reduced by the addition of glutathione to the incubation mixture. In addition to the covalently bound metabolites, water-soluble metabolites derived from arachidonic acid metabolism were also observed. The amount of water-soluble metabolites was small in each tissue except for the rat stomach fundus. In the rat stomach fundus the water-soluble metabolites accounted for over 50% of the total metabolites. Conditions which would tend to increase or decrease the levels of free prostaglandin endoperoxides during the incubation of arachidonic acid with the microsomes gave increased or decreased levels of covalent binding. Our data suggest that the prostaglandin endoperoxides are responsible for the covalent binding observed during prostaglandin biosynthesis. This covalent binding to tissue macromolecules may be of physiological and pathological significance.     

Activation mechanism of prostaglandin endoperoxide synthetase by hemoproteins

The mechanism of the activation of prostaglandin endoperoxide synthetase by hemeproteins was investigated using the enzyme purified from bovine seminal vesicle microsomes. At pH 8, the maximal enzyme activities with methemoglobin (2 microM), indoleamine 2,3-dioxygenase (2 microM), and metmyoglobin (2 microM) were 70%, 42%, and 15% of that with 1 microM hematin. Apomyoglobin and apohemoglobin inhibited the enzyme activities caused by hemoproteins as well as that caused by hematin. The inhibition was removed by the addition of excess hematin. The dissociation of heme from hemoproteins was demonstrated by trapping the free heme with human albumin or to a DE-52 column. The dissociation of heme from methemoglobin was facilitated by increasing concentrations of arachidonic acid. The amount of heme dissociated from hemoproteins (methemoglobin, metmyoglobin, and indoleamine 2,3-dioxygenase) in the presence of arachidonic acid correlated with their stimulatory effects on the prostaglandin endoperoxide synthetase activity. Horseradish peroxidase and beef liver catalase, the hemes of which were not dissociated in the presence of arachidonic acid, were ineffective in activating prostaglandin endoperoxide synthetase. Spectrophotometric titration of prostaglandin endoperoxide synthetase with hematin demonstrated that the enzyme bound hematin at the ratio of 1 mol/mol with an association constant of 0.6 x 10(8) M-1. From these results, we conclude that hemoproteins themselves are ineffective in activating prostaglandin endoperoxide synthetase and free hematin dissociated from the hemoproteins by the interaction of arachidonic acid is the activating factor for the enzyme.     

Prostaglandin synthetase activity from human rheumatoid synovial tissue and its inhibition by non-steroidal anti-inflammatory drugs.

1. Prostaglandin synthetase activity was found in a microsomal fraction from human rheumatoid synovia. 2. The microsomes produced PGE2 and a small amount of PGF2 when incubated with arachidonic acid. 3. The pH optimum of the enzyme from this source was similar to that found with microsomal preparations from rabbit renal medullae and bovine seminal vesicles. 4. The enzyme was inhibited in vitro by the non-steroidal anti-inflammatory drugs flurbiprofen, indomethacin and aspirin in the same rank order of potency as prostaglandin synthetase from other tissues.     

Peroxidatic oxidation of benzo[a]pyrene and prostaglandin biosynthesis.

The arachidonic acid dependent oxidation of benzo[a]pyrene to a mixture of 3,6-, 1,6-, and 6,12-quinones has been studied by using enzyme preparations from sheep seminal vesicles. Maximal oxidation is observed at 100 microM benzo[a]pyrene and 150 microM arachidonic acid. The arachidonic acid dependent oxidation is peroxidatic and utilizes prostaglandin G2 (PGG2), generated in situ from arachidonate, as the hydroperoxide substrate. 15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid is equivalent to PGG2 as a hydroperoxide substrate, but hydrogen peroxide, cumene hydroperoxide, and tert-butyl hydroperoxide are much poorer substrates. Arachidonic acid dependent benzo[a]pyrene oxidation by microsomal and solubilized enzyme preparations is markedly.     

Some characteristics of the prostaglandin synthesizing system in rabbit kidney microsomes.

The prostaglandin synthesizing system in rabbit kidney microsomes was characterised using a radiometric assay. Three prostaglandins (F2alpha, E2 and D2) were formed form (1-14C)arachidonic acid, a small amount of prostaglandin A2 was also detected but this was formed non-enzymatically. Biosynthesis was stimulated by reduced-glutathione and 1-adrenaline and was inhibited by aspirin-like drugs. The enzyme system was sensitive to small changes in pH. There were substantial differences in drug sensitivity and optimal reaction conditions between this prostaglandin synthesizing system and the one from bovine seminal vesicles.     

The oxidation of arachidonic acid by the cyclooxygenase activity of purified prostaglandin H synthase: spin trapping of a carbon-centered free radical intermediate

The ESR spin trapping technique was used to study the first detectable radical intermediate in the oxidation of arachidonic acid by purified prostaglandin H synthase. The holoenzyme and the apoenzyme, reconstituted with either hematin or Mn2+ protoporphyrin IX, were investigated. Depending on the different types of enzyme activity present, arachidonic acid was oxidized to at least two free radicals. One of these radicals is thought to be the first ESR detectable radical intermediate in the conversion of arachidonic acid to prostaglandin G2 and was detected previously in incubations of ram seminal vesicle microsomes, which are rich in prostaglandin H synthase. The ESR findings correlated with oxygen incorporation into arachidonic acid and prostaglandin formation, where the spin trap inhibits oxygen incorporation and prostaglandin formation by apparently competing with oxygen for the carbon-centered radical. Substitution of arachidonic acid by octadeuterated (5, 6, 8, 9, 11, 12, 14, 15)-arachidonic acid confirmed that the radical adduct contained arachidonic acid that is bound to the spin trap at one of these eight positions. An attempt was made to explain the apparent time lag between the metabolic activity observed in the oxygraph measurements and the appearance of the trapped radical signals.     

Prostaglandin synthase-catalyzed metabolic activation of some aromatic amines to genotoxic products

Cultured human fibroblasts were incubated with different aromatic amines in the presence of different activation systems and the induction of strand breaks in fibroblast DNA was studied. In the presence of ram seminal vesicle microsomes and arachidonic acid, DNA strand breaks were induced by 2-naphthylamine, 2,4-diaminotoluene and 4-methoxy-m-phenylenediamine. This effect was decreased when the prostaglandin synthase of the ram seminal vesicle microsomes was inhibited. The data suggest that metabolic activation catalyzed by prostaglandin synthase may be of importance in the formation of genotoxic products by certain urinary tract carcinogens.     

Solubilization and partial purification of prostaglandin endoperoxide synthetase of rabbit kidney medulla.

The microsomes of rabbit kidney medulla converted arachidonic acid into prostaglandin E2 in the presence of hemoglobin, tryptophan and glutathione as activators. When themicrosomal suspension was treated with 1% Tween 20, a solubilized enzyme was obtained which catalyzed the conversion of arachidonic acid to prostaglandins G2 and H2. The solubilized enzyme was adsorbed to and then eluted from an omega-aminooctyl Sepharose 4B column, resulting in about 10-fold purification over the microsomes. The partially purified enzyme produced predominantly prostaglandin G2 in the presence of hemoglobin, while prostaglandin H2 was produced in the presence of both hemoglobin and tryptophan. The stimulation of prostaglandin endoperoxide formation was also observed with other heme and aromatic compounds. Prostaglandin H2 synthesis was inhibited by a variety of compounds including non-steroidal anti-inflammatory drugs, thiol compounds and prostaglandin analogues with a thiol group(s).     

Acetylation of the NH2-terminal serine of prostaglandin synthetase by aspirin.

Aspirin (acetylsalicylic acid) inhibits prostaglandin synthesis by acetylating an active site portion of the enzyme, prostaglandin synthetase. In the current study, the site of acetylation has been demonstrated to be a seryl residue at the NH2 terminus of the enzyme. Purified [3H]acetyl enzyme was prepared from seminal vesicle homogenates treated with [acetyl-3H]aspirin. The [3H]acetate to protein bond was stable to hydroxylamine, indicating an N-acetyl linkage. The [3H]acetyl enzyme was fragmented sequentially with cyanogen bromide, trypsin, and pronase. The 3H material isolated from the pronase digest was identified as N-acetylserine. This finding indicates that the oxygenase portion of prostaglandin synthetase has an NH2-terminal serine which is involved in enzymatic activity and is susceptible to acetylation by aspirin.     

A carbon-centered free radical intermediate in the prostaglandin synthetase oxidation of arachidonic acid. Spin trapping and oxygen uptake studies

The ESR spin-trapping technique has been used to identify a free radical involved in the oxygenation of arachidonic acid by ram seminal vesicle microsomes. The ESR spectrum of the radical adduct indicates that a carbon-centered arachidonic acid free radical has been observed. The formation of this species is inhibited by indomethacin, but not by phenol, and it is probably the first intermediate formed during the prostaglandin synthetase-catalyzed oxidation of arachidonic acid. The chemical identity of the trapped radical was substantiated with an independent synthesis of a closely related radical adduct.     

Flurbiprofen: highly potent inhibitor of prostaglandin synthesis.

Flurbiprofen, 2-(2-fluoro-4-biphenylyl)propionic acid, inhibited the formation of prostaglandin E2 from arachidonic acid by bovine seminal vesicular microsomes. It was found that flurbiprofen was an approx. 12.5-fold better inhibitor than indomethacin by comparison of their I50 values. It was suggested that the inhibition of prostaglandin synthesis by flurbiprofen might be due to the inhibition of the endoperoxygenase which catalyzed conversion of arachidonic acid to cyclic endoperoxide. Other carboxylic acid compounds such as aspirin, ibuprofen and indomethacin showed the same type of inhibition as flurbiprofen. In contrast, phenylbutazone which was a pyrozolone derivative inhibited the formation of prostaglandin E2, but not affected the endoperoxygenase reaction. The kinetic studies for inhibition of prostaglandin E2 synthetase indicated that flurbiprofen competitively inhibited prostaglandin E2 synthesis, just like indomethacin. The Ki values were estimated to be 0.128 micron for flurbiprofen and 3.18 micron for indomethacin.     

Metabolism of 3-methylindole by prostaglandin H synthase in ram seminal vesicles

3-Methylindole (3MI) causes a highly tissue- and species-selective lesion of the lung. Metabolic activation of 3MI by the NADPH-dependent mixed function oxidase (MFO) system is the initial event in the lung-specific toxicity. One-electron co-oxidation of 3MI by prostaglandin H synthase (PHS) has been implicated as an alternative mechanism for toxicity in the lung that contains high PHS activity. The objective of this study was to determine if 3MI can be co-oxidized by the arachidonic acid dependent PHS complex. Ram seminal vesicle (RSV) microsomes, which lack MFO activity, were used as a source of PHS. Incubations of RSV microsomes with 3MI, at a concentration as low as 0.01 mM, showed an increase in PHS activity, as indicated by an enhanced rate of oxygen consumption. This effect was arachidonic acid dependent and was inhibited (98%) by indomethacin. Addition of 3MI resulted in a concentration-dependent increase in PHS-catalyzed prostaglandin biosynthesis from [14C]arachidonic acid. PHS-dependent oxidative metabolism of [14C]3MI resulted in a twofold increase in ethyl acetate extracted radiolabelled metabolites. ESR spin-trapping studies demonstrated the presence of a 3MI free radical generated from the metabolism of 3MI by horseradish peroxidase, a model system of PHS hydroperoxidase. The results indicate that 3MI can be co-oxidized by the arachidonic acid-dependent PHS complex. Co-oxidation of 3MI by PHS may play a role in the tissue specificity of 3MI-induced pneumotoxicity.     

Prostaglandin synthesis inhibited by formamidine pesticides.

The formamidine pesticides, chlordimeform and amitraz, were shown to have both antipyretic and anti-inflammatory activity when given intraperitoneally to rats at 5 to 80 mg per kg. They reduced yeast-induced fever in rats with potencies intermediate between those of indomethacin and aspirin, and antagonized the carageenin-induced swelling of the hind paw. In both these actions, chlordimeform was more potent than amitraz. Both formamidines also inhibited the synthesis of prostaglandin E₂ from arachidonic acid by bovine seminal vesicle microsomes. At an arachidonic acid concentration of 0.4 μM, the I₅₀ values for chlordimeform and amitraz were 34 and 880 μM respectively, compared to 0.4 μM and 790 μM for indomethacin and aspirin. These aspirin-like actions may provide a clue to some of the physiological effects of the formamidines, which represent a new and unsual group of prostaglandin synthetase inhibitors.     

Spectral intermediates of prostaglandin hydroperoxidase

Microsomes from ram seminal vesicles or purified prostaglandin H synthase supplemented with either arachidonic acid or prostaglandin G2 formed an unstable spectral intermediate with maxima at 430 nm, 525 nm and 555 nm and minima at 410 nm, 490 nm and 630 nm. At -15 degrees C the band at 430 nm disappeared within 4 min whereas the trough at 410 nm increased three fold. At higher temperatures (10-37 degrees C) spectral complex formation and decay were observed in less than 1 s. An apparent KS-value of about 3 microM was determined for the titration of purified prostaglandin synthase with prostaglandin G2 at -20 degrees C. Substrates for cooxidation reactions of prostaglandin synthase such as phenol, hydroquinone and reduced glutathione as well as the peroxidase inhibitors cyanide and azide inhibited the prostaglandin G2-induced spectral complex formation. The oxene donor iodosobenzene and hydrogen peroxide formed a spectral intermediate analogous to the complex observed with prostaglandin G2 or arachidonic acid in ram seminal vesicle microsomes as well as with the purified prostaglandin synthase. These results are interpreted as the formation of a ferryl-oxo complex (FeO)3+ of the heme of prostaglandin synthase with prostaglandin G2 analogous to the formation of compound I of horseradish peroxidase.     

Selective inhibition of prostaglandin endoperoxide thromboxane isomerase by 1-carboxyalkylimidazoles

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

Effect of polyamines on prostaglandin synthesis in various cell-free systems

Synthesis of PGF_2α by bovine uterus and guinea pig lung microsomes and that of TXB₂ by human platelet and rat spleen microsomes were stimulated by spermine. PGE₂ synthesis by bovine seminal vesicle and porcine lung microsomes, and 6-keto-PGF_1α synthesis by bovine seminal vesicle and uterus microsomes were inhibited by spermine. When phospholipid-free prostaglandin synthetase from bovine seminal vesicle was used instead of microsomes, the inhibition of PGE₂ synthesis by spermine disappeared. The inhibition of PGE₂ synthesis by spermine gradually appeared with an increase of phospholipid added. Among phospholipids tested, phosphatidylcholine was the most effective for the inhibition of PGE₂ synthesis by spermine.     

Endogenous inhibition of arachidonic acid metabolism in the endometrium of the sheep

Arachidonic acid is metabolised via the cyclo-oxygenase pathway to several biologically active metabolites. These metabolites control important reproductive functions like luteolysis of the corpus luteum. The metabolism of arachidonic acid was studied by the enzymatic conversion of [1-14C]-labelled arachidonic acid in sheep endometrial tissue. The inhibitory capacity of sheep endometrial tissue was measured by the enzymatic conversion of [1-14C]-arachidonic acid by sheep seminal vesicular gland microsomes. Endometrial microsomes converted arachidonic acid into different prostaglandins and monohydroxy acids but at a low rate. A factor(s) inhibiting both prostaglandin and monohydroxy acid synthesis was found in both the microsomal and cytosolic fractions of endometrial tissue. A very high inhibitory potency of prostaglandin and monohydroxy acid synthesis, calculated as IC50 values, was found in cytosolic fractions. For comparison IC50 values of indomethacin, mefenamic acid, carprofen and acetylsalicylic acid were also calculated in this in vitro system. These data indicate that both prostaglandin and monohydroxy acid synthesizing capacities and an inhibitory factor(s) are present in sheep endometrium and possibly regulate arachidonic acid metabolism in this tissue.     

Effect of castration on prostaglandin-mediated changes in membrane potential and prostaglandin synthesis in guinea-pig seminal vesicle tissue

Arachidonic acid stimulated an increase in transmural electrical potential difference (p.d.) in guinea-pig seminal vesicle tissue in vitro. Pretreatment with indomethacin abolished this response. Indomethacin pretreatment did not prevent the p.d. from increasing in response to theophylline. Changes in p.d. in response to arachidonic acid were greatly attenuated, and the response to theophylline was abolished in seminal vesicle tissue taken from castrated guinea-pigs. Seminal vesicles, aorta and ileum taken from castrated guinea-pigs synthesized and released more prostaglandins than did those from control animals. It is concluded that the effects of arachidonic acid on p.d. are mediated by its metabolism to prostaglandins; the inability of seminal vesicles from castrated animals to respond to arachidonic acid is not a result of a decrease in prostaglandin production, but is more likely a result of other degenerative changes attendant upon castration; and androgens appear to have some regulatory function on prostaglandin synthesis in a variety of tissues.