Indomethacin and aspirin inhibition of prostaglandin E2 synthesis by sheep seminal vesicles microsome powder

Sheep seminal vesicles microsome powder was used as a source of prostaglandin synthetase in studies on the nature of inhibition of prostaglandin synthesis by indomethacin and aspirin. Irdomethacin inhibition was found to be highly irreversible, although partial recovery of synthetase activity was obtained after extensive dialysis. A major difference was observed between the effects of aspirin and indomethacin on prostaglandin synthetase activity in seminal vesicles slices. Enzyme activity in microsomes prepared from slices incubated with aspirin was markedly inhibited while the activity in microsomes prepared after incubation with indomethacin was not affected. These results suggest that indomethacin may penetrate intracellularly very slowly, or not at all, and raise a question as to whether the inhibition by indomethacin $\text{in vivo}$ is mediated via direct inhibition of prostaglandin synthesis.     

Metabolism of paracetamol to a glutathione conjugate catalyzed by prostaglandin synthetase

Microsomes isolated from sheep seminal vesicles (SSV) were found to catalyze the metabolic activation of paracetamol as evidenced by rapid formation of paracetamol glutathione conjugate when SSV microsomes were incubated with paracetamol in the presence of arachidonic acid and GSH. The activity was inhibited by indomethacin indicating the involvement of prostaglandin synthetase in the reaction. The initial activity was very rapid, and the affinity for paracetamol in the reaction was high, since formation of the glutathione conjugate was optimal already at 0.2 mM drug concentration.It is concluded that the activation of paracetamol is due to the peroxidase activity of prostaglandin synthetase in SSV microsomes, since linolenic acid hydroperoxide was also able to support the reaction.     

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.     

Prostaglandin synthetase activity of goat vesicular microsomes: Co-factor requirement and effect of calcium ions

The effects of several co-factors and bivalent cations on the activity of prostaglandin synthetase isolated from goat seminal vesicles were studied. Ca²⁺ appears to play a regulatory role in the biosynthesis of prostaglandin E₂ by goat vesicular microsomes as the normal parabolic time course of synthesis changed to a sigmoid curve in the presence of 4 mM Ca²⁺ and to nearly a hyperbolic pattern when the microsomes were preincubated with the metal ions. The Ca²⁺ modulated reaction showed increased rate of prostaglandin E₂ synthesis only when the period of incubation was extended beyond 30 min. The co-factor requirement of the goat enzyme was similar to that of the bovine and ovine prostaglandin synthetase systems.     

On the mode of action and biochemical properties of anti-inflammatory drugs-II.

The inhibition of prostaglandin (PG) synthetase by nonsteroidal anti-inflammatory drugs (NSAID) is not well understood. Co-factors (glutathione and hydroquinone) are needed for maximum enzymatic activity in vitro, and we suggest that NSAID might inhibit PG synthetase partly by interfering with co-factor induced stimulation of the enzyme. This hypothesis was tested by: A) Examining the effect of glutathione, noradrenaline and hydroquinone on bull seminal vesicle (BSV) PG synthetase in vitro. The stimulatory effects were concentration-dependent. B) Three structurally distinct NSAID, indomethacin, aspirin and paracetamol, inhibited the stimulation by each co-factor in a concentration-related manner. Drug effectiveness also depended on the concentration of co-factor.     

On the mode of action and biochemical properties of anti-inflammatory drugs-II

The inhibition of prostaglandin (PG) synthetase by nonsteroidal anti-inflammatory drugs (NSAID) is not well understood. Co-factors (glutathione and hydroquinone) are needed for maximum enzymatic activity $\text{in vitro}$, and we suggest that NSAID might inhibit PG synthetase partly by interfering with co-factor induced stimulation of the enzyme. This hypothesis was tested by:A) Examining the effect of glutathione, noradrenaline and hydroquinone on bull seminal vesicle (BSV) PG synthetase $\text{in vitro}$. The stimulatory effects were concentration-dependent.B) Three structurally distinct NSAID, indomethacin, aspirin and paracetamol, inhibited the stimulation by each co-factor in a concentration-related manner. Drug effectiveness also depended on the concentration of co-factor.     

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.     

Biochemical characterization of prostaglandin 19-hydroxylase of seminal vesicles

The microsomal fraction of homogenates of seminal vesicles of men and monkeys, Macaca fascicularis, were analyzed for prostaglandin (PG) 19-hydroxylase activity. The microsomes of the monkey seminal vesicles, supplemented with 1 mM NADPH, metabolized 0.2 mM PGE1 to 19-hydroxy-PGE1 at a mean rate of 0.26 nmol/min/mg of protein (with an apparent Km and an apparent Vmax of 40 microM and 0.30 nmol/min/mg of protein, respectively). The enzyme catalyzed the incorporation of atmospheric oxygen into the substrate. Substituting NADH for NADPH reduced the prostaglandin E1 19-hydroxylase activity to 40%. Carbon monoxide and proadifen (SKF 525A) inhibited the enzyme. Prostaglandin E2 (0.2 mM) was metabolized to 19-hydroxyprostaglandin E2 (0.2 nmol/min/mg of protein), but PGE1 was preferred as a substrate. Prostaglandin B1 was metabolized to 18-hydroxy-, 19-hydroxy-, and 20-hydroxyprostaglandin B1 at a combined rate of approximately 25% of prostaglandin E1. 19-Hydroxyprostaglandin B1 was the main product. The microsomes of human seminal vesicles metabolized 0.2 mM PGE2 to 19-hydroxy-PGE2 in the presence of 1 mM NADPH, while carbon monoxide inhibited this reaction. These results suggest that prostaglandin 19-hydroxylase of seminal vesicles might be a cytochrome P-450. The biosynthesis of 19-hydroxyprostaglandin E1 and 19-hydroxyprostaglandin E2 was also studied in vivo in man by analysis of the product/substrate ratios (i.e. 19-hydroxyprostaglandin E1/prostaglandin E1 and 19-hydroxyprostaglandin E2/prostaglandin E2) in a series of consecutive ejaculates, which were obtained during short intervals. There was a 10-fold interindividual difference in these ratios. Although the product/substrate ratios decreased, the 19-hydroxylation of E prostaglandins appeared to be efficient in vivo, which was in contrast to the rather slow biosynthesis in vitro.     

A specific prostaglandin I2 synthetase inhibitor, 3-hydroperoxy-3-methyl-2-phenyl-3H-indole.

Inhibitory effects of 3-hydroperoxy-3-methyl-2-phenyl-3H-indole(HPI) on prostaglandin endoperoxide synthase(EC 1.14.99.1) and prostaglandin I₂(PGI₂) synthetase were compared with those of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid, namely, 15-hydroperoxyarachidonic acid(15-HPAA) and tranylcypromine (TCP). Sheep seminal vesicle microsomes were used as a source of prostaglandin endoperoxide synthase and bovine aortic microsomes as that of PGI₂ synthetase. 15-HPAA and HPI inhibited PGI₂ synthetase with IC₅₀s of 5 × 10^?7 and 3.5 × 10^?6 M, respectively, whereas neither compound had effect on prostaglandin endoperoxide synthase at the concentration inhibiting PGI₂ synthetase by 90%. TCP was a weak(IC₅₀ = 5 × 10^?4M) PGI₂ synthetase inhibitor with low specificity.     

Flurbiprofen-Sepharose chromatography of the prostaglandin synthetase from bovine seminal vesicles

Flurbiprofen-Sepharose and Acetyl-Sepharose have been prepared by coupling dl-2-(2-fluoro-4-biphenylyl)propionic acid [Flurbiprofen] and acetic acid, respectively, to 3-(N-[3-aminopropyl])aminopropyl Sepharose 4B using a water soluble carbodiimide. The arachidonic acid oxygenase activity of solubilized bovine seminal vesicle microsomes is retarded during chromatography on Flurbiprofen-Sepharose but not Acetyl-Sepharose. Thus binding of the oxygenase to Flurbiprofen-Sepharose results from interaction with the immobilized inhibitor. However, the impure oxygenase is either not bound and/or not eluted in a biospecific manner since the abilities of flufenamic acid, R(+) and S(-)-5-cyclohexylindan-1-carnboxylic acid, and R and S-Naproxen to remove the enzyme from Flurbiprofen-Sepharose do not parallel the relative efficacies of these compounds as prostaglandin synthesis inhibitors. Nevertheless, gradient elution of arachidonic acid oxygenase activity from Flurbiprofen-Sepharose with flufenamic acid provides a 15 fold enrichment of the enzyme from solubilized bovine seminal vesicle microsomes in 80% yield indicating that this chromatographic reagent can be a powerful tool for use in purification of the prostaglandin synthetase.     

Prostaglandin biosynthesis and its regulation in the bovine endometrium: A comparison between nonpregnant and pregnant status

Prostaglandin F(2alpha) (PGF(2alpha)), an arachidonic acid metabolism product of the prostaglandin synthetase pathway, is synthesized and released from the endometrium during luteolysis in nonpregnant animals. When proper conception occurs, the synthesis and release pattern is changed to maintain the corpus luteum (CL) function. The biosynthesis of prostaglandins in the bovine endometrium was highest in the microsomes but of low order. In nonpregnancy, the formation of prostaglandins from labelled precursor acid was higher than in pregnancy. Besides the prostaglandin synthetase, an inhibiting activity on the conversion of arachidonic acid to prostaglandins was found in both the nonpregnant and pregnant endometrium. During luteolysis (Day 17), a low inhibiting capacity was seen in comparison with other days of the estrous cycle (Days 1, 4 and 14). The inhibitory capacity was very high on Days 16 to 20, 25, and 31 of pregnancy. In the nonpregnant endometrium at Day 17, a very low inhibitor potency, calculated as IC(50) values, was found both in the cytoplasma and in the microsomes, whereas during early pregnancy (Days 17, 18, and 20) both cytoplasma and microsomes possessed very high inhibitor potency. This finding indicates that the bovine endometrium contains both prostaglandin synthetase and an unknown potent inhibitor of prostaglandin biosynthesis that regulates prostaglandin biosynthesis both during the estrous cycle and early pregnancy.     

A test for the intermediacy of 11-hydroperoxyeicosa-5,8,12,14-tetraenoic acid [11-HPETE] in prostaglandin biosynthesis

11-Hydroperoxy-eicosa-5,8,12,14-tetraenoic acid [11-HPETE] was prepared by chromatographic separation of the hydroperoxides formed from the singlet oxygen oxidation of arachidonic acid [20:4]. 1-[¹⁴C]-HPETE was incubated with prostaglandin endoperoxide synthetase preparations from ram seminal vesicles. No prostaglandins products deriving from 11-HPETE were detected in any of the incubations. 11-Hydroxy-eicosa-5,8,12,14-tetraenoic acid [11-HETE], formed by the action of the hydroperoxidase component of prostaglandin endoperoxidase synthetase was the major product formed. The peroxidase activity was absolutely dependent on epinephrine and was stimulated by hematin. 11-HPETE does not appreciably effect the extent of conversion of arachidonic acid into prostaglandin.     

Acid and neutral alpha-glucosidase in the reproductive organs and seminal plasma of the bull

A synthetic substrate (p-nitrophenyl-alpha-D-glucopyranoside) was used to measure the acid and neutral alpha-glucosidase activity in bull seminal plasma, spermatozoa and in homogenates of bull reproductive organs. Marked differences were observed in the activities of these enzymes in the various tissues studied. Epididymis and particularly its caput region contained the highest specific activity of acid alpha-glucosidase. The activity of neutral alpha-glucosidase was highest in testis and in different parts of the epididymis. Seminal plasma, spermatozoa and seminal vesicle secretion contained only the acid enzyme activity. After fractionation with anion exchange chromatography in HPLC (Mono Q) and chromatofocussing, acid alpha-glucosidase activity of seminal plasma was recovered in two fractions with different pI values. The corresponding activities were found in the secretion of seminal vesicles, which thus form the major secretory source of seminal plasma acid alpha-glucosidase. In the fractionation with gel filtration on Sepharose 6B, the acid alpha-glucosidase had a smaller molecular weight than did the neutral enzyme. In anion exchange chromatography and chromatofocussing the testicular and epididymal homogenates each contained two acid and two neutral isoenzymes. In both fractionations the elution pattern of acid alpha-glucosidase was clearly different from that of the enzymes in seminal plasma. The pH optimum of acid alpha-glucosidase ranged from 3.75 to 4.5 and that of the neutral enzyme from 6.5 to 7.0. The neutral activity was more sensitive to many divalent metal ions and differences were also observed in the response of the enzymes to different concentrations of turanose and KCl.     

Biosynthesis of 18(RD)-hydroxyeicosatetraenoic acid from arachidonic acid by microsomes of monkey seminal vesicles. Some properties of a novel fatty acid omega 3-hydroxylase and omega 3-epoxygenase

Microsomes of seminal vesicles of the cynomolgus monkey were incubated with [14C]5,8,11,14-eicosatetraenoic (arachidonic) acid and NADPH for 40 min at 37 degrees C and the products were characterized. Prostaglandins F2 alpha and E2 were the two main metabolites (approximately 52% of radioactivity), while 18(R)-hydroxy-cis-5,8,11,14-eicosatetraenoic acid (18(R)-HETE) was identified as the main, less polar product (approximately 13%). Significant biosynthesis of the 19-hydroxy or 20-hydroxy metabolites of arachidonic acid could not be detected. The formation of 18(R)-HETE was further investigated in the presence of a prostaglandin synthesis inhibitor, diclofenac sodium. The omega 3-hydroxylation was only partly supported by substituting NADH for NADPH. The hydroxyl oxygen of 18(R)-HETE was derived from the atmosphere and the omega 3-hydroxylation was inhibited by proadifen and partly inhibited by carbon monoxide. These findings suggest that 18(R)-HETE is formed by a cytochrome P-450 (P-450 omega 3). Linoleic acid and 8,11,14-eicosatetraenoic acid were also substrates of the enzyme, but stearic acid was not metabolized. 5,8,11,14,17-Eicosatetraenoic acid was oxygenated under these conditions mainly to 17,18-dihydroxy-5,8,11,14-eicosatetraenoic acid, presumably formed from 17(18)-epoxy-5,8,11,14-eicosatetraenoic acid by hydrolysis. The seminal microsomes thus seem to possess both omega 3-hydroxylase and omega 3-epoxygenase activity. These seminal vesicles also contain prostaglandin E 19-hydroxylase (Oliw, E.H., Kinn, A.-C., and Kvist, U. (1988) J. Biol. Chem. 263, 7222-7227). The presence of arachidonate omega 3-hydroxylase and prostaglandin E 19-hydroxylase was assessed in microsomes of adult and juvenile monkey livers. Arachidonic acid was metabolized extensively to diols (via epoxides), but 18-HETE could not be detected. In contrast, prostaglandin E1 was slowly hydroxylated mainly to 19-hydroxyprostaglandin E1 by both adult male and female juvenile hepatic microsomes. The results indicate that P-450 omega 3 of seminal vesicles might be a tissue-specific enzyme.     

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.     

In vitro and ex vivo effects of antidepressants on rat brain membrane-bound phosphatidylinositol synthetase activity

The in vitro and ex vivo effects of antidepressant drugs on membrane-bound phosphatidylinositol (PI) synthetase and PI: myo-inositol exchange enzyme activities were examined. In rat brain subcellular fractions, PI synthetase occurred exclusively in the microsomes. In comparison, the activity of CDP-diglyceride independent PI: myo-inositol exchange enzyme was low (3%). Of the various CDP-diglycerides tested for the activation of PI synthetase, CDP-dipalmitin was the most active. Addition of 1 mM of desipramine, amitriptyline, imipramine, iprindole, clomipramine and mianserin in vitro significantly inhibited (30–60%) PI synthetase activity, whereas the same concentration of zimelidine and fluoxetine had no effect. At low liponucleotide concentrations, PI synthetase activity was significantly enhanced by imipramine (1 mM), whereas the enzyme activity was inhibited at higher liponucleotide concentrations (>0.3 mM). In contrast, imipramine had no effect on the PI: myo-inositol exchange enzyme activity. No significant alteration in the PI synthetase activity was found following either acute (2 h) or chronic (21 d) treatment of rats with imipramine. The above results indicate that the de novo synthesis of PI is inhibited in vitro but not ex vivo by some antidepressant drugs. However, in view of the high concentration of the drugs required, the pharmacological significance of this inhibitory action with respect to their therapeutic effects is doubtful.     

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.     

The modulation of prostaglandin synthesis by peritoneal fluids

Biological fluids from several sources (e.g. blood, fetal urine, amniotic fluid) have been shown to contain factors that modulate prostaglandin (PG) synthesis. In this study, we investigated the possibility that peritoneal fluid contains substances that may regulate PG synthesis. Peritoneal fluids were obtained from women undergoing diagnostic laparoscopy for infertility. Fluids from women without evident pelvic pathology were incubated with prostaglandin synthase prepared from bull seminal vesicles in the presence of excess arachidonic acid, and the production of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha was quantified by specific radioimmunoassay. The untreated fluids inhibited potently the synthesis of PGE2 but such inhibitory activity was not extractable by chloroform:methanol. An ultrafiltrate of the fluid containing molecules smaller than 10,000 Daltons stimulated PGF2 alpha synthesis but this activity was also lost after extraction. The extracted fluid did, however, stimulate the synthesis of prostacyclin (as reflected by 6-keto-PGF1 alpha).