Characterization of the biosynthetic pathway of prostaglandin D2 in human platelet-rich plasma

The biosynthetic mechanism of prostaglandin D2 in human platelet-rich plasma has been investigated. Platelet-rich plasma was separated into washed platelets and platelet-poor plasma, and [1-14C]prostaglandin H2 was incubated with each fraction. The enzymatic conversion of the endoperoxide to prostaglandin D2 was found only in platelet-poor plasma and not in washed platelets or platelet lysate. This prostaglandin D synthetase activity was purified to homogeneity and identified as serum albumin by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focusing, and immunoelectrophoresis. The optimal pH and Km value for prostaglandin H2 were 9.0 and 6 microM, respectively. Glutathione was not required for the activity. Although prostaglandin H2 ws converted to prostaglandin D2 and E2 in the reaction, only the prostaglandin D2 formation was dependent on the protein amount and abolished by prior boiling. The action of this activity under physiological conditions was examined in a model system constituted of serum albumin and washed platelets. Prostaglandin D2 formation was observed in association with thrombin-evoked platelet aggregation in this system and was proportional to the number of platelets and the concentration of serum albumin, suggesting that thrombin-stimulated platelets released prostaglandin H2, and the latter compound was then converted to prostaglandin D2 by the action of serum albumin. Consistent with this interpretation, prostaglandin H2 added to platelet-rich plasma was converted in part to prostaglandin D2, and the aggregation caused by this endoperoxide was greatly enhanced by neutralizing the action of prostaglandin D2 with anti-prostaglandin D2 antiserum.     

Mechanism of prostaglandin biosynthesis in rabbit kidney medulla. A rate-limiting step and the differential stimulatory actions of L-adrenaline and glutathione.

Microsomal prostaglandin synthase (EC 1.14.99.1) from rabbit kidney medulla was assayed with [5,6,8,9,11,12,14,15-3H]-and [1-14C]-arachidonic acid as the substrate. The ratios of prostaglandin F2 alpha to prostaglandin E2 and to prostaglandin D2 were determined by both 3H and 14C labelling. When 3H was used as a label the ratios were much higher than with 14C labelling indicating that the removal of hydrogen at C-9 or C-11 was the rate-limiting step in the biosynthesis of prostaglandin E2 or prostaglandin D2. This finding shows that the octatritiated arachidonic acid is not the appropriate substrate marker for studying the regulation of the synthesis of different prostaglandins by various agents. When the enzyme assay was carried out in the presence of SnCL2, which was capable of accumulating exclusively prostaglandin F2alpha at the expenses of prostaglandin E2 and prostaglandin D2, the addition of L-adrenaline to the microsomal fraction either alone or with reduced glutathione equally stimulated the formation of prostaglandin F2alpha, whereas the addition of reduced glutathione to the microsomal fraction either alone or with L-adrenaline produced no additional effect. These results suggest that endoperoxide is formed as the common intermediate for the biosynthesis of three different prostaglandins in rabbit kidney medulla, and that L-adrenaline stimulates the synthesis of endoperoxide, whereas reduced glutathione facilitates the formation of prostaglandins from endoperoxide.     

Interaction of prostaglandin E2 with human serum albumin

Using equilibrium dialysis, protein fluorescence and fluorescent probing as well as chemical modification, the interaction of prostaglandin E2 with human serum albumin was studied. The serum albumin molecule has a highly specific prostaglandin E2-binding site. The binding of prostaglandin causes conformational rearrangements in the protein molecule. The amino group of serum albumin is involved in the interaction with prostaglandin E2. Prolonged exposure of prostaglandin E2 to serum albumin causes partial irreversible binding of prostaglandin molecules to the protein.     

Development of a radioimmunoassay for prostaglandin D2 using an antiserum against 11-methoxime prostaglandin D2

A sensitive and specific radioimmunoassay for prostaglandin D2 has been developed using its stabilized 11-methoxime derivative, which was obtained after treatment of prostaglandin D2 with methoxamine-HCl. The antiserum was obtained after injection of prostaglandin D2-methoxamine coupled to bovine serum albumin. A (125I)-Histamide prostaglandin D2-methoxamine tracer was prepared by iodination of the corresponding histamide, followed by thin layer chromatography purification. The sensitivity of the assay was 280 femtomoles per ml at 50% displacement. The cross reactivities were 15% with prostaglandin D1-methoxamine and less than 0.20% with other prostaglandins. Determination of the half-life of prostaglandin D2 in a solution containing albumin was also carried out, since it has been shown to catalyze prostaglandin D2 destruction. The unstability of this prostaglandin is due to the presence of a beta-hydroxy ketone group, and all prostaglandins possessing this labile moiety could be stabilized by such a derivatization before developing a radioimmunoassay.     

Platelet rich plasma transforms exogenous prostaglandin endoperoxide H2 into thromboxane A2

Platelet rich plasma transforms exogenous prostaglandin endoperoxide H2 into thromboxane A2 immediately prior to the initiation of irreversible aggregation. Selective thromboxane synthetase inhibitors block thromboxane A2 formation and aggregation. Thromboxane A2 formation appears to be essential during arachidonate mediated aggregation. The results presented reconcile the previously accepted paradoxical behavior of thromboxane synthetase in platelet rich plasma toward the prostaglandin endoperoxide H2 substrate.     

On the interrelationship of prostaglandin endoperoxide G2 and cyclic nucleotides in platelet function.

The prostaglandin endoperoxide G2 caused rapid aggregation and relase of ADP and [14C]serotonin in human platelets. Since the presence of the ADP phosphorylating system creatine phosphate/creatine phosphokinase markedly inhibited the aggregation caused by the endoperoxide, this effect seemed to be mediated mainly by ADP, which is instantaneously released by the endoperoxide. The prostaglandin G2 counteracted the increasing effect of prostaglandin E1 on the adenosine 3':5'-monophosphate (cAMP) levels in platelet-rich plasma. This effect of prostaglandin G2 was only observed when ADP was released by the endoperoxide. This finding indicates that the effect of prostaglandin G2 on the cAMP levels in platelet-rich plasma is principally mediated by ADP. The rapid release of ADP by prostaglandin G2 and the time courses for the effects of the endoperoxide and ADP on the level of cAMP give further evidence for this hypothesis. ADP also caused primary aggregation in the presence of indomethacin, and prostaglandin synthesis inhibitors did not influence the decreasing effect of ADP on the cAMP levels. N2,O2-Dibutyrylguanosine 3':5'-monophosphate did not influence the aggregation and release-reaction caused by ADP and no changes of the cGMP levels were observed after addition of prostaglandin G2.     

Influence of dietary vitamin E on prostaglandin biosynthesis in rat blood.

A vitamin E (alpha-tocopherol) deficient diet stimulated prostaglandin biosynthesis in coagulating rat blood. Prostaglandins were extracted from serum, purified and bioassayed. The identity of prostaglandin E2 was confirmed by gas chromatography-mass spectrometry. Withholding vitamin E from the diet caused a marked increase in PGE2 and a lesser increase in PGF2alpha production in serum. In rats maintained on diets containing different concentrations of vitamin E, serum concentrations of PGE2 and PGF2alpha were inversely related to serum concentrations of alpha-tocopherol. These data suggest that in vitro alpha-tocopherol inhibits the endogenous conversion of arachidonic acid into PGE2 and PGF2alpha. The possibility that alpha-tocopherol may inhibit the formation of endoperoxide intermediates of PGE2 and PGF2alpha biosynthesis and subsequent induction of platelet aggregation is discussed.     

Effects of prostaglandin E2, 16,16-dimethyl prostaglandin E2 and a prostaglandin endoperoxide analogue (U-46619) on gastric secretory volume, [H+] and mucus synthesis and secretion in the rat

The effects of orally administered prostaglandin E2, 16,16-dimethyl prostaglandin E2 and U-46619, an analogue of the prostaglandin endoperoxide PGH2, on gastric secretory volume, acid and mucus were studied in the rat. All of the compounds significantly increased the volume of gastric secretion, mucus secretion, measured as N-acetylneuraminic acid and mucus synthesis measured as the incorporation of [3H]-glucosamine into mucosal glycoprotein; however, only PGE2 and 16,16-dimethyl PGE2 inhibited acid secretion. U-46619, 1.5 mg/kg provided significant protection against ethanol-induced gastric ulcers, an effect that has been previously shown for the other two compounds. These studies provide additional evidence that prostaglandin induced mucosal protection may be related to an effect on mucus and on stimulation of nonparietal cell gastric secretion. Further study of these parameters may be important in the development of antiulcer drugs for long term clinical use.     

Effect of organic sulfur compounds on the chemical and enzymatic transformations of prostaglandin endoperoxide H2.

The effects of several sulfur organic compounds on the enzymatic and non-enzymatic transformations of prostaglandin endoperoxide H2 to prostaglandins were studied. Mercaptoethanol, methional alpha-lipoic acid and dimercaptopropanol increased the chemical (i.e. non-enzymatic) reduction of prostaglandin H2 to prostaglandin F2alpha but except for alpha-lipoic acid, had no effect on the enzymatic conversion of prostaglandin H2 to prostaglandin. In contrast, reduced glutathione showed no effect on the chemical conversion of prostaglandin H2, but exerted a marked and specific stimulation on the enzymatic isomerization of prostaglandin H2 to prostaglandin E2. This specific effect of gluthione may serve to regulate the overall intracellular activity of prostaglandin synthetase as well as the particular ratio of prostaglandins produced.     

Amino acid substitutions in the human glutathione S-transferases confer different specificities in the prostaglandin endoperoxide conversion pathway

The human glutathione S-transferases 1-1 and 2-2, which differ from each other by 11 amino acids, have different catalytic activities against cumene hydroperoxide and t-butyl hydroperoxide. Using prostaglandin H2 as the peroxide substrate, we found that GSH S-transferase 1-1 catalyzed the transformation of prostaglandin H2 to prostaglandin F2 alpha and E2 at a 4:1 ratio whereas GSH S-transferase 2-2 produced primarily prostaglandin D2 and F2 alpha at a 4:1 ratio. Our results indicate that GSH S-transferases catalyze the reduction and isomerization of prostaglandin H2 endoperoxide in vitro. We suggest that the amino acid substitutions between these two isozymes may be responsible for the difference in catalytic specificities. We propose that these isozymes are important reagents for the biosynthesis of various prostaglandins.     

Regulation of 3T3-L1 fibroblast differentiation by prostacyclin (prostaglandin I2)

Prostaglandin biosynthesis and prostaglandin-stimulated cyclic AMP accumulation were studied in 3T3-L1 fibroblasts as they differentiated into adipocytes. Incubation of 3T3-L1 membranes with [1-14C]prostaglandin H2, and subsequent radio-TLC analysis, showed that prostacyclin (prostaglandin I2) is the principal enzymatically synthesized prostaglandin in this cell line. Confirmation of the radiochemical data was obtained by demonstrating the presence of 6-keto-prostaglandin F1 alpha, the stable hydrolysis product of prostaglandin I2, by gas chromatography-mass spectrometry. In support of previous work, indomethacin, the prostaglandin endoperoxide synthetase (EC 1.14.99.1) inhibitor, accelerated 3T3-L1 differentiation. More importantly, the incubation of 3T3-L1 cells with insulin and the prostaglandin I2 synthetase inhibitor 9,11-azoprosta-5,13-dienoic acid (azo analog I) also enhanced the rate of cellular differentiation, even though this compound does not inhibit the synthesis of other prostaglandins. The repeated addition of exogenous prostaglandin I2 to 3T3-L1 cells inhibited insulin- and indomethacin-mediated differentiation. When 3T3-L1 cells were exposed to various prostaglandins and the cyclic AMP levels were measured, prostaglandin I2 proved to be the most potent stimulator of cyclic AMP accumulation, followed by prostaglandin E1 greater than prostaglandin H2 much greater than prostaglandin E2, while prostaglandin D2 was inactive. As 3T3-L1 cells differentiate, the ability of prostaglandin I2 or prostaglandin H2 to stimulate cyclic AMP accumulation progressively diminishes. It is suggested that 3T3-L1 differentiation may be controlled by the rate of prostaglandin I2 synthesis and/or sensitivity of the adenylate cyclase to prostaglandin I2.     

Purification and properties of prostaglandin D synthetase from rat brain.

The prostaglandin D synthetase system was isolated from rat brain. Prostaglandin endoperoxide synthetase solubilized from a microsomal fraction catalyzed the conversion of arachidonic acid to prostaglandin H2 in the presence of heme and tryptophan. Prostaglandin D synthetase (prostaglandin endoperoxidase-D isomerase) catalyzing the isomerization of prostaglandin H2 to prostaglandin D2 was found predominantly in a cytosol fraction and was purified to apparent homogeneity with a specific activity of 1.7 mumol/min/mg of protein at 24 degrees C. The enzyme also acted upon prostaglandin G2 and produced a compound presumed to be 15-hydroperoxy-prostaglandin D2. Glutathione was not required for the enzyme reaction, but the enzyme was stabilized by thiol compounds including glutathione. The enzyme was inhibited by p-chloromercuribenzoic acid in a reversible manner. The purified enzyme was essentially free of the glutathione S-transferase activity which was found in the cytosol of brain.     

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).     

Influence of dietary vitamin E on prostaglandin biosynthesis in rat blood

A vitamin E (-tocopherol) deficient diet stimulated prostaglandin biosynthesis in coagulating rat blood. Prostaglandins were extracted from serum, purified and bioassayed. The identity of prostaglandin E₂ was confirmed by gas chromatography-mass spectrometry. Withholding vitamin E from the diet caused a marked increase in PGE₂ and a lesser increase in PGF₂ production in serum. In rats maintained on diets containing different concentrations of vitamin E, serum concentrations of PGE₂ and PGF₂ were inversely related to serum concentrations of -tocopherol. These data suggest that in vivo -tocopherol inhibits the endogenous conversion of arachidonic acid into PGE₂ and PGF₂. The possibility that -tocopherol may inhibit the formation of endoperoxide intermediates of PGE₂ and PGF₂ biosynthesis and subsequent induction of platelet aggregation is discussed.     

Kinetic studies on the conversion of prostaglandin endoperoxide PGH2 by thromboxane synthase

We have investigated the time course of formation of thromboxane A₂, thromboxane B₂, and the C-17 hydroxy fatty acid, HHT, from arachidonic acid in a washed human platelet suspension. Our results indicate that HHT is not a breakdown product of thromboxane A₂, but rather thromboxane A₂ decomposes exclusively into thromboxane B₂. The kinetics of formation of thromboxane B₂ from the endoperoxide prostaglandin H₂ in human platelet microsomes was examined. Our data suggest that a bimolecular reaction is involved in the formation of thromboxane A₂ from prostaglandin H₂ and that thromboxane synthase is not an isomerase, but may be acting via a dismutase-type reaction. One possibility is that thromboxane and HHT are produced simultaneously from two molecules of prostaglandin H₂.     

HTRF-based assay for microsomal prostaglandin E2 synthase-1 activity

Microsomal prostaglandin E(2) synthase-1 (mPGES-1) catalyzes the formation of prostaglandin E(2) (PGE(2)) from the endoperoxide prostaglandin H( 2) (PGH(2)). Expression of this enzyme is induced during the inflammatory response, and mouse knockout experiments suggest it may be an attractive target for antiarthritic therapies. Assaying the activity of this enzyme in vitro is challenging because of the unstable nature of the PGH( 2) substrate. Here, the authors present an mPGES-1 activity assay suitable for characterization of enzyme preparations and for determining the potency of inhibitor compounds. This plate-based competition assay uses homogenous time-resolved fluorescence to measure PGE(2) produced by the enzyme. The assay is insensitive to DMSO concentration up to 10% and does not require extensive washes after the initial enzyme reaction is concluded, making it a simple and convenient way to assess mPGES-1 inhibition.     

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.     

Stimulatory effect of prostaglandin E2 on 1 alpha,25-dihydroxyvitamin D3 synthesis in rats.

The effect of prostaglandin E2 on accumulation in plasma of 1 alpha,25-dihydroxy[3H]vitamin D3 from 25-hydroxy[3H]vitamin D3 was studied in vivo using vitamin D-deficient thyroparathyroidectomized rats. Intra-arterial infusion of 10-50 micrograms of prostaglandin E2/h caused a significant stimulation of 1 alpha,25-dihydroxy[3H]vitamin D3 production. No significant changes in plasma Ca2+ and Pi concentrations or urinary cyclic AMP excretion were observed after prostaglandin E2 infusion. Theophylline did not enhance the effect of a submaximal dose of prostaglandin E2 on 1 alpha,25-dihydroxy[3H]vitamin D3 production. These data indicate that prostaglandin E2 stimulates plasma accumulation of 1 alpha,25-dihydroxy[3H]vitamin D3 independent of the adenylate cyclase/cyclic AMP system, and suggest that prostaglandin E2 has a modulatory role in the regulation of 25-hydroxyvitamin D3 1 alpha-hydroxylase in the kidney.     

Dexamethasone stimulates arachidonic acid conversion to prostaglandin E2 in human amnion cells

The purpose of this investigation was to study the mechanism of stimulation of PGE2 output from human amnion epithelial cells by the synthetic glucocorticoid dexamethasone. Cells incubated in serum-free pseudo-amniotic fluid produced very low levels of PGE2, even when arachidonic acid (1 microM) was present. Pretreatment of cells with dexamethasone (50 nM) for 21 h increased the PGE2 output 6- to 7-fold in 2-h incubations only in the presence of arachidonic acid. The RNA synthesis inhibitor, actinomycin D (1 microgram/ml), and the protein synthesis inhibitor, cycloheximide (40 micrograms/ml), each blocked dexamethasone-stimulated arachidonic acid conversion to PGE2. The time course of these events suggests that dexamethasone first initiates RNA synthesis. Acetylsalicylic acid, a specific and irreversible blocker of prostaglandin endoperoxide H synthase (cyclooxygenase), was used to determine whether dexamethasone could stimulate new enzyme synthesis. Cells treated first with acetylsalicylic acid (30 min) then dexamethasone (22 h) produced as much PGE2 in response to 1 microM arachidonate as did cells exposed to dexamethasone only. Exposing cells to acetylsalicylic acid after dexamethasone completely eliminated PGE2 output. These data suggest that dexamethasone stimulates the synthesis of prostaglandin endoperoxide H synthase.     

Elimination of low steady-state concentrations of E15,6-3H2]prostaglandin E1 in the pulmonary and the systemic circulations of anaesthetized rats

The elimination of [3H]prostaglandin E1 in anaesthetized rats was studied by continuous intravenous or intraarterial infusions, producing steady-state concentrations at the level of endogenous prostaglandin E2 in mixed venous blood. Blood samples (0.5 ml) were collected from the carotid artery or the right atrium, respectively. The levels of [3H]prostaglandin E1 were measured at different infusion time intervals and the 3H-labeled hydrophobic metabolites characterized. Cardiac output was estimated by a modification of the dye injection method, using 125I-labelled albumin as the marker. From the cardiac output and the rate of infusion, the fractional clearance of the lung and the systemic beds in the steady-state situation were estimated to 88.3 +/- 3.2% and 54.1 +/- 15.2% (mean +/- S.D.), RESPECTIVELY. The hydrophobic metabolites were characterized chromatographically on Sephadez LH-20 columns, using synthetically prepared [14C]prostaglandin metabolites as internal standards and markers. The identities of some metabolites were further established by derivative formation to a constant [3H]/[14C] ratio. The major metabolite was 15-keto-13,14-dihydro-[3H]prostaglandin E1, while 15-keto-[3H]prostaglandin E1 and 13,14-dihydro-[3H]prostaglandin E1 could not be demonstrated.