PGD2 formation in the vasculature: Characteristics of rat tail vein prostaglandin endorperoxide-D isomerase

Rat tail vein homogenates, microsome and high speed supernatant fractions were incubated with [1-¹⁴C]prostaglandin endoperoxide (PGH₂) and products separated and identified by radio-thinlayer chromatography. PGI₂ synthase was localized to the microsomal fraction, but exhibited low activity compared to rat tail arteries prepared in the same manner. PGH-D isomerase was maximally active in the presence of reduced glutathione at pH 7.5–8.0, exhibited a K_m for PGH₂ of 33 μM, and was inhibited sulfhydryl-directed reagents. The similarities of this enzyme to PGD synthase of the rat cerebral microvasculature are discussed.     

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.     

Prostaglandin D synthase in microvessels from the rat cerebral cortex

Microvessels, a mixture composed predominantly of small arterioles and capillaries (7-80 micro diameter), were isolated from the rat cerebral cortex by selective nylon sieving and glass bead elutriation. The morphology and purity of the microvessel and cerebral cortex filtrate (virtually free of vascular contamination) were monitored by light microscopy and by the activity of several enzymes: gamma -glutamyl transpeptidase, GSH-S-transferase, prostacyclin synthase and PGD synthase. Prostacyclin and PGD synthesizing activities as well as gamma-glutamyl transpeptidase activity were localized to the microvessels of the rat cerebral cortex whereas GSH-S-Transferase was restricted to the non-vascular filtrate fraction. The characteristics of the PGD synthase were similar to those of the purified enzyme previously described for the rat brain. The microvessel (MV) PGD synthase was localized to the cytosol fraction of the microvessels and did not require reduced glutathione for activity. The enzyme was inhibited by pre-incubation with p-hydroxymercuribenzoate (ImM) or N-ethylmaleimide (ImM). The MV RGD synthase saturated at 15-20 microM PGH2, exhibited an apparent Km of 9.6 microM, and a pH optimum of 8.0-8.1. These findings suggest roles for both prostacyclin and PGD synthesis by the rat cerebral vasculature in the autoregulation of cerebral blood flow and/or neural function. These studies also indicate that the major source of PGI2 and PGD2 synthesis by rat brain homogenates is the microvasculature.     

Epidermal growth factor stimulation of prostaglandin E2 biosynthesis in amnion cells. Induction of prostaglandin H2 synthase

Amnion is believed to be a tissue of signal importance, anatomically and functionally, in the maintenance of pregnancy and during the initiation of parturition. Epidermal growth factor (EGF)-like agents cause a striking increase in the secretion of prostaglandin E2 (PGE2) in human amnion cells but only if arachidonic acid is present in the culture medium. To investigate the regulation of arachidonic acid metabolism by EGF-like agents in amnion, we used mEGF and human amnion cells in primary monolayer culture as a model system. The amount of PGE2 secreted into the culture medium was quantified by radioimmunoassay and the rate of conversion of [14C]arachidonic acid to [14C]PGE2 (PGH2 synthase activity) in cell sonicates was determined under optimal in vitro conditions. Treatment of amnion cells with mEGF led to a marked increase in the rate of production of PGE2. The specific activity of PGH2 synthase (viz. the combined activities of prostaglandin endoperoxide (PGH2) synthase and PGH2-PGE isomerase) was increased by 2-5-fold in cells treated with mEGF. Treatment of amnion cells with mEGF for 4 h did not affect the specific activities of phospholipase A2 or phosphatidylinositol-specific phospholipase C. By immunoisolation of newly synthesized, [35S]methionine-labeled PGH2 synthase, we found that mEGF stimulated de novo synthesis of the enzyme. Thus, mEGF acts in human amnion cells in primary monolayer culture to increase the rate of PGE2 biosynthesis by a mechanism that involves induction of PGH2 synthase; the manifestation of EGF action on PGE2 biosynthesis is dependent on the presence of nonesterified arachidonic acid.     

Ovarian prostaglandin endoperoxide synthase: cellular localization during the rat estrous cycle

The specific cellular localization of prostaglandin endoperoxide (PGH) synthase was studied throughout the rat estrous cycle. Animals were necropsied at 1300 h on each day of the 4-day cycle, and an additional group was necropsied at 2300 h on proestrus. Ovaries were removed and processed for cellular identification of PGH synthase by immunohistochemistry. At all stages of the cycle, intense immunostaining was observed in newly formed corpora lutea. Luteal cells were immunoreactive, but the connective tissue centrum was unstained. Interstitial tissue contained heavily labeled cells, whereas the germinal epithelium exhibited faint staining. During estrus, metestrus, and diestrus, thecal cells from preantral and antral follicles contained PGH synthase immunoreactivity, but granulosa cells were unstained. Faint staining of mural granulosa cells was observed first in 78% of preovulatory follicles (less than 400-microns diameter) in ovaries collected on the afternoon of proestrus. After the luteinizing hormone surge, 95% of the preovulatory follicles exhibited PGH synthase staining. The percentage of immunoreactive granulosa cells in these preovulatory follicles increased 4-fold in ovaries collected at 2300 h on proestrus. The presence of ovarian PGH synthase throughout the rat estrous cycle and the changes in cellular localization may reflect the potential role of PGs in follicular and luteal function.     

Stereoselectivity of the epoxidation of 7,8-dihydrobenzo[a]pyrene by prostaglandin H synthase and cytochrome P-450 determined by the identification of polyguanylic acid adducts

The stereoselectivity of the oxidation of 7,8-dihydrobenzo[a]pyrene (H2BP) to 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (H4BP-epoxide) by prostaglandin H (PGH) synthase and cytochrome P-450 has been studied using microsomal preparations from ram seminal vesicles and rat liver. Incubations were performed in the presence of polyguanylic acid and the adducts formed between H4BP-epoxide and guanosine were isolated following the recovery and hydrolysis of the poly(G). When (+/-)-H4BP-epoxide was reacted with poly(G), four diastereomeric adducts were formed by the cis and trans addition of the exocyclic amino group of guanine to the benzylic carbon of the epoxide enantiomers. Each diastereomer was identified by a combination of ultraviolet, nuclear magnetic resonance, circular dichroism, and mass spectroscopy. Under comparable conditions, ram seminal vesicle microsomes in the presence of arachidonic acid triggered the binding of H2BP to poly(G) to a greater extent than rat liver microsomes from untreated and phenobarbital- and methylcholanthrene pretreated animals in the presence of NADPH. Quantitation of the (-)-cis- and (+)-cis-guanosine adducts revealed the degree of stereoselectivity of epoxidation. The ratio of (-)/(+) adducts was 54:46 for PGH synthase and 89:11 (control), 62:38 (phenobarbital), and 69:31 (methylcholanthrene) for cytochrome P-450-catalyzed reactions. PGH synthase catalyzed the epoxidation of H2BP with little or no stereoselectivity in contrast to cytochrome P-450. The utility of the poly(G) binding technique for the elucidation of the stereoselective generation of chiral electrophiles is discussed along with the mechanistic implications of the results.     

Inhibition of GSH-dependent PGH2 isomerase in mammary adenocarcinoma cells by allicin

We have reported that allicin, a constituent of garlic oil, has no effect on the activities of platelet cyclooxygenase or thromboxane synthase, or vascular PGI2 synthase. The effect of allicin on glutathione (GSH) dependent PGH2 to PGE2 isomerase is unknown. We therefore studied the effect of allicin on PGE2 biosynthesis in a murine mammary adenocarcinoma cell line (No 4526). Intact or sonicated cells were incubated with either 14C-arachidonic acid (AA) or 14C-PGH2, respectively. Following metabolism, products were extracted, separated by TLC and analyzed by radiochromatographic scan. PGE2 was predominantly formed with minimal amounts of PGF2 alpha and PGD2. Formation of 6-keto-PGF1 alpha or TXB2 was not detected indicating the absence of TXA2 and PGI2 synthase activity. Indomethacin and ibuprofen inhibited the PGE2 formation (p less than 0.05). The enzymatic PGE2 formation in sonicates was blocked by depletion of the cellular non-protein thiols by buthionine sulfoximine and was shown to be dependent on GSH. Allicin, over the range of 10-1000 microM, inhibited the formation of PGE2 in cells exposed to 2.0 microM 14C-AA for 20 min. and in sonicated cells incubated with 20.0 microM 14C-PGH2 for 2 min (p less than 0.05). Allicin did not alter cyclooxygenase-mediated oxygen utilization in ram seminal vessicle microsomes, suggesting that allicin selectively inhibits the GSH-dependent PGH2 to PGE2 isomerase in this adenocarcinoma cell line.     

Cellular localization of ovarian prostaglandin endoperoxide synthase during pseudopregnancy in the rat

The specific cellular localization of prostaglandin endoperoxide (PGH) synthase, the enzyme responsible for initiating the conversion of arachidonic acid to prostaglandins, was studied throughout pseudopregnancy in the rat. Pseudopregnancy was induced by administration of eCG (20 IU) to immature, 27-day-old rats followed by hCG injection (10 IU) on Day 29. Animals were necropsied on Days 1 (Day 1 = 1 day post hCG), 5, 9, and 13 of pseudopregnancy. Ovaries were removed and processed for cellular identification of PGH synthase by immunohistochemistry. Immunoreactive PGH synthase was distributed throughout the CL at each of the 4 different days of pseudopregnancy, with the majority of the luteal cells exhibiting varying degrees of staining. The connective tissue centrum of the CL, however, lacked PGH synthase immunoreactivity. Quantitative assessment of the immunostaining distribution was accomplished with a computer-based image analysis program (Kontron IBAS). Results are expressed as the percentage of a digitized luteal area that contained intense immunoreactivity between Day 1 (0.6 +/- 0.2% immunoreactive area) and Day 5 (16.8 +/- 2.7%) of pseudopregnancy. The area of luteal immunostaining was similar on Day 5 and Day 9 (16.8 +/- 2.7% and 14.7 +/- 2.0%, respectively) followed by a decrease (p less than 0.05) in immunoreactivity on Day 13 (9.1 +/- 2.2%). The region of the CL adjacent to the germinal epithelium had an increase (p less than 0.01) in PG synthase staining distribution compared to the region of the CL adjacent to the ovarian medulla on all days of pseudopregnancy except Day 1. These findings demonstrate that PGH synthase is present in the rat CL throughout pseudopregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical localization of prostaglandin H synthase in the embryo and uterus of the mouse from ovulation through implantation

Prostaglandins (PGs) in the embryo and endometrium are involved in processes that are important for implantation. Although the presence of PGs (PGE2, PGF2 alpha, PGI2) in decidualized endometrium has been widely reported, less is known about the capacity of the pre-implantation embryo to synthesize PGs. Prostaglandin H (PGH) synthase is necessary for the production of PGs. Using an immunohistochemical method, PGH synthase was localized in the mouse embryo and uterus from superovulation through embryo implantation. No PGH synthase was detected in oocytes at the time of ovulation or in single-cell embryos 1 day post-fertilization (PF). Circular areas of immunostaining became evident in the cytoplasm of blastomeres at the morula stage (day 3 PF). After implantation (day 5 PF), a low level of PGH synthase reactivity was observed in embryonic cells; no PGH synthase was detected in the embryo by day 7 PF. The endometrial glands exhibited maximal immunostaining by day 3 PF, and after implantation, PGH synthase appeared in decidual cells along the border of placentation. Low levels of PGH synthase reactivity were detected in myometrial cells during the period after superovulation through day 7 PF. This is the first demonstration of PGH synthase in the mouse embryo prior to apposition with glandular endometrial epithelium, supporting the hypothesis that the embryo has the potential to produce PGs that may mediate autocrine and/or paracrine responses at the time of nidation.     

Products, kinetics, and substrate specificity of homogeneous thromboxane synthase from human platelets: development of a novel enzyme assay

Homogeneous thromboxane synthase from human platelets converted prostaglandin H2 (PGH2) to thromboxane A2 (measured as thromboxane B2, TxB2), 12(L)-hydroxy-5,8,10-heptadecatrienoic acid (HHT), and malondialdehyde (MDA) in equimolar amounts under a variety of experimental conditions. PGG2 was transformed to MDA and corresponding 15- and 12-hydroperoxy products. PGH1 was enzymatically transformed into 12(L)-hydroxy-8,10-heptadecadienoic acid (HHD) and PGH3 into TxB3 and 12(L)-hydroxy-5,8,10,14-heptadecatetraenoic acid (delta 14-HHT) as earlier reported for solubilized and partially purified thromboxane synthase preparations. The ratio of thromboxane to C17 hydroxy fatty acid formation was 1:1 with PGG2, PGH2, and PGH3 as substrates. These results confirm and extend earlier observations with partially purified enzyme that the three products are formed in a common enzymatic pathway (Diczfalusy, U., Falardeau, P., and Hammarstr?m, S. (1977) FEBS Lett. 84, 271-274). A convenient spectrophotometric assay for thromboxane synthase activity measuring the ultraviolet light absorption of the C17 hydroxy acid formed (e.g., HHT) was developed. The validity of the assay was determined employing specific inhibitors for thromboxane synthase. The substrate specificity of thromboxane synthase was determined using this assay. PGG2 and PGH3 showed Vmax and KM values similar to those of PGH2. The KM value of PGH1 was also identical to that of PGH2 but the Vmax value PGH1 was more than twice as high as that of PGH2.     

Cloning, expression, and up-regulation of inducible rat prostaglandin e synthase during lipopolysaccharide-induced pyresis and adjuvant-induced arthritis

We have cloned and expressed the inducible form of prostaglandin (PG) E synthase from rat and characterized its regulation of expression in several tissues after in vivo lipopoylsaccharide (LPS) challenge. The rat PGE synthase is 80% identical to the human enzyme at the amino acid level and catalyzes the conversion of PGH(2) to PGE(2) when overexpressed in Chinese hamster ovary K1 (CHO-K1) cells. PGE synthase activity was measured using [(3)H]PGH(2) as substrate and stannous chloride to terminate the reaction and convert all unreacted unstable PGH(2) to PGF(2alpha) before high pressure liquid chromatography analysis. We assessed the induction of PGE synthase in tissues from Harlan Sprague-Dawley rats after LPS-induced pyresis in vivo. Rat PGE synthase was up-regulated at the mRNA level in lung, colon, brain, heart, testis, spleen, and seminal vesicles. Cyclooxygenase (COX)-2 and interleukin 1beta were also up-regulated in these tissues, although to different extents than PGE synthase. PGE synthase and COX-2 were also up-regulated to the greatest extent in a rat model of adjuvant-induced arthritis. The RNA induction of PGE synthase in lung and the adjuvant-treated paw correlated with a 3.8- and 16-fold induction of protein seen in these tissues by immunoblot analysis. Because PGE synthase is a member of the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family, of which leukotriene (LT) C(4) synthase and 5-lipoxygenase-activating protein are also members, we tested the effect of LTC(4) and the 5-lipoxygenase-activating protein inhibitor MK-886 on PGE synthase activity. LTC(4) and MK-886 were found to inhibit the activity with IC(50) values of 1.2 and 3.2 microm, respectively. The results demonstrate that PGE synthase is up-regulated in vivo after LPS or adjuvant administration and suggest that this is a key enzyme involved in the formation of PGE(2) in COX-2-mediated inflammatory and pyretic responses.     

Concentration-activity profile of the modulation of cyclooxygenase product formation by reduced glutathione in microsomal fractions from the goat lung

Age-related changes in pulmonary formation of arachidonic acid (AA) metabolites are thought to play an important role in regulating cardiopulmonary function. This study addresses the potential role of reduced glutathione (GSH) in modulating cyclooxygenase product formation in the developing lung. Prostaglandin H2 (PGH2) metabolism was studied in microsomal fractions isolated from the lungs of unventilated fetal, neonatal and adult goats. GSH-dependent PGH2 to PGE2 isomerase activity in microsomal fractions from the perinatal (fetal and neonatal) goat lung was not saturable with respect to GSH and can respond to changes in GSH concentration over the range of 0.01 to 30 mM, which encompasses the full range the intracellular GSH levels reported in the literature. However, in fractions from the adult, a lower rate of PGE2 formation is observed at higher GSH concentrations. In addition, the tissue levels of GSH exhibited developmental stage-related differences with fetal being higher than neonatal or adult. The present observations may have physiologic relevance, in that decreases in pulmonary GSH levels after birth may contribute to decreases in plasma PGE2 levels by decreasing pulmonary PGE2 synthesis, thereby contributing to closure of the ductus arteriosus; conversely, increased GSH levels associated with hyperoxia may contribute to persistence of ductal patency. Formation of 6-keto-PGF1 alpha and of TXB2 (the stable metabolites of prostacyclin and TXA2) was decreased when PGE2 formation was increased by GSH activation of PGE2 isomerase in fractions isolated from all three developmental stages. A similar pattern of product formation was observed when AA was employed as substrate. These data suggest the possibility that changes in GSH concentration may modulate eicosanoid formation in cells that contain GSH-dependent PGE2 isomerase, as well as either or both prostacyclin or thromboxane synthase(s).     

Biphasic effects of nonsteroidal anti-inflammatory drugs on prostaglandin production by cultured rat calvariae

We have studied the effects on bone of three structurally dissimilar non-steroidal anti-inflammatory drugs which inhibit prostaglandin cyclo-oxygenase activity (PGH synthase); indomethacin, flurbiprofen, and piroxicam. We used cultures of half calvaria from neonatal or fetal rats to measure effects on PGE2 production, measured by radioimmunoassay. In four day neonatal rat calvaria, indomethacin inhibited PGE2 release into the medium by 80% at 10(-8) M, while flurbiprofen and piroxicam produced similar inhibition at 10(-6) M. However, at 10(-10) M, treatment with all three compounds resulted in an increase in medium PGE2 concentration of 60 to 120%. To assess the mechanism of this effect, bones were labeled with [3H]-arachidonic acid, washed and cultured in the presence or absence of piroxicam. At 10(-6) M, piroxicam inhibited production of cyclo-oxygenase products and arachidonic acid release. However, at 10(-10) M, there was a substantial increase in labeled products, particularly PGE2, despite a further decrease in arachidonic acid release. In 21 day fetal rat cultures, flurbiprofen was found to increase PGE2 release both in control cultures and cultures which had been incubated with cortisol (10(-8) M) to reduce endogenous arachidonic acid release and supplied with exogenous arachidonic acid (10(-5) M) to provide a substrate. These results indicate that three potent inhibitors of PGH synthase can, paradoxically, increase prostaglandin production at low concentrations. The effect does not appear to be due to increased arachidonic acid release, and could be due to increased PGH synthase activity.     

Controlled tryptic digestion of prostaglandin H synthase. Characterization of protein fragments and enhanced rate of proteolysis of oxidatively inactivated enzyme

Treatment of prostaglandin H (PGH) synthase (70 kDa) with trypsin generates fragments of 33 and 38 kDa. Each of the fragments was purified by reverse-phase high performance liquid chromatography (HPLC) using acetonitrile/water/trifluoroacetic acid gradients. Amino acid sequence analysis indicates that the 33-kDa protein contains the NH2 terminus of PGH synthase. Neither the 33- nor 38-kDa fragment isolated by HPLC exhibits any PGH synthase activity; however, cleavage of intact enzyme to 33- and 38-kDa fragments to the extent of 90% only reduces cyclooxygenase activity by 40%. This implies that the cleaved proteins or a complex formed between them retains the conformation necessary for enzyme activity. Extensive attempts to resolve active fragments from each other or from intact enzyme were unsuccessful; intact enzyme and digestion fragments cochromatograph under all conditions employed. Treatment of PGH synthase with [3H]acetylsalicylic acid followed by trypsin digestion introduces [3H]acetyl moieties into the intact protein and the 38-kDa fragment (0.8-0.9 acetyl group/subunit). Nearly complete conversion of PGH synthase to 33- and 38-kDa fragments by exposure to high concentrations of trypsin prior to [3H]acetylsalicylic acid treatment results in labeling of the 38-kDa fragment, but not the 33-kDa fragment. The present findings are consistent with the presence of a membrane-binding domain (33 kDa) and an active site domain (38 kDa) in the 70-kDa subunit of PGH synthase. They also suggest that, following cleavage, the 38-kDa fragment retains the structural features responsible for the cyclooxygenase activity and selective aspirin labeling of PGH synthase. PGH synthase undergoes self-catalyzed inactivation by oxidants generated during its catalytic turnover. When PGH synthase, inactivated by treatment with arachidonic acid or hydrogen peroxide, was treated with trypsin it was cleaved two to three times faster than unoxidized enzyme. Addition of heme to oxidized PGH synthase did not reconstitute cyclooxygenase activity or resistance to trypsin cleavage. Spectrophotometric studies demonstrated that oxidatively inactivated enzyme did not bind heme. This implies that oxidation of protein residues as well as the heme prosthetic group is an important determinant of proteolytic sensitivity. Oxidative modification may mark PGH synthase for proteolytic cleavage and turnover.     

Mechanism for antiplatelet effect of onion: AA release inhibition, thromboxane A(2)synthase inhibition and TXA(2)/PGH(2)receptor blockade

Antiplatelet actions of aqueous extract of onion were investigated in rat and human platelet. IC(50)values of onion extract for collagen-, thrombin-, arachidonic acid (AA)-induced aggregations and collagen-induced thromboxane A(2)(TXA(2)) formation were 0.17 +/- 0. 01, 0.23 + 0.03, 0.34 +/- 0.02 and 0.12 +/- 0.01 g/ml, respectively. [(3)H]-AA release induced by collagen (10 microg/ml) in rat platelet was decreased by onion compared to control (22.1 +/- 2.13 and 5.2 +/- 0.82% of total [(3)H]-AA incorporated, respectively). In fura-2 loaded platelets, the elevation of intracellular Ca(2+)concentration stimulated by collagen was inhibited by onion. Onion had no cytotoxic effect in platelet. Onion significantly inhibited TXA(2)synthase activity without influence on COX activity. Platelet aggregation induced by U46619, a stable TXA(2)mimetic, was inhibited by onion, indicating its antagonism for TXA(2)/PGH(2)receptor. These results suggest that the mechanism for antiplatelet effect of onion may, at least partly, involve AA release diminution, TXA(2)synthase inhibition and TXA(2)/PGH(2)receptor blockade.     

Control of prostacyclin synthesis in pregnancy-induced hypertension

Uterine prostacyclin synthase (PGI synthase) and prostaglandin endoperoxide synthase (PGH synthase) concentrations, measured by specific immunoradiometric assays, did not differ between patients with severe pregnancy-induced hypertension (syn. pre-eclampsia; n = 5) and normotensive gravidae (n = 6) with comparable gestational ages (34 - 38 weeks). Myometrial microsomes from pre-eclamptic women contained ten times more PGI synthase than PGH synthase and the ratio of PGI synthase to PGH synthase (mean +/- SD; 10.1 +/- 3.9) was not different from that in normotensive pregnancies. None of the pre-eclamptic patients had myometrial enzyme levels that were more than one standard deviation below the mean established previously for pregnancies ranging from 32 to 42 weeks of gestation. These findings indicate that the commonly observed association between deficient PGI2 production and pregnancy-induced hypertension cannot be explained in terms of a generalized lack of immunoassayable prostacyclin or prostaglandin endoperoxide synthases.     

Essential histidines of prostaglandin endoperoxide synthase. His-309 is involved in heme binding

Prostaglandin endoperoxide (PGH) synthase has a single iron protoporphyrin IX which is required for both the cyclooxygenase and peroxidase activities of the enzyme. At room temperature, the heme iron is coordinated at the axial position by an imidazole, and about 20% of the heme iron is coordinated at the distal position by an imidazole. We have used site-directed mutagenesis to investigate which histidine residues are involved in PGH synthase catalysis and heme binding. Individual mutant cDNAs for ovine PGH synthases were prepared with amino acid substitutions at each of 13 conserved histidines. cos-1 cells were transfected with each of these cDNAs, and the cyclooxygenase and peroxidase activities of the resulting microsomal PGH synthases were measured. Mutant PGH synthases in which His-207, His-309, or His-388 was replaced with either glutamine or alanine lacked both activities. Gln-386 and Ala-386 PGH synthase mutants exhibited cyclooxygenase but not peroxidase activities. Other mutants exhibited both activities at varying levels. Because binding of heme renders native PGh synthase resistant to cleavage by trypsin, we examined the effects of heme on the relative sensitivities of native, Ala-204, Ala-207, Ala-309, Ala-386, and Ala-388 mutant PGH synthases to trypsin as a measure of the heme-protein interaction. The Ala-309 PGh synthase mutant was notably hypersensitive to tryptic cleavage, even in the presence of exogenous heme; in contrast, the native enzyme and the other alanine mutants exhibited similar, lower sensitivities toward trypsin and, except for the Ala-386 mutant, were partially protected from trypsin cleavage by heme. Preincubation of the native and each of the alanine mutant PGH synthases, including the Ala-309 mutant, with indomethacin protected the proteins from trypsin cleavage. Thus, all the mutant proteins retain sufficient three-dimensional structure to bind cyclooxygenase inhibitors. Our results suggest that His-309 is one of the heme ligands, probably the axial ligand, of PGH synthase. Two other histidines, His-207 and His-388, are essential for both PGH synthase activities suggesting that either His-207 or His-388 can serve as the distal heme ligand; however, the trypsin cleavage measurements imply that neither His-207 nor His-388 is required for heme binding. This is consistent with the fact that only 20% of the distal coordination position of the heme iron of PGH synthase is occupied by an imidazole side chain.     

The N-terminal membrane anchor domain of the membrane-bound prostacyclin synthase involved in the substrate presentation of the coupling reaction with cyclooxygenase

To mimic the native conditions, the cyclooxygenase (COX)/prostaglandin I(2) synthase (PGIS) coupling reaction system was used to determine the coordination of PGIS with COX for the biosynthesis of prostacyclin (PGI(2)) using arachidonic acid (AA) as a substrate in a membrane-bound environment. The membrane-bound PGIS exhibited a faster isomerization of PGH(2) produced by COX to PGI(2) than the detergent-solubilized PGIS. To determine whether the N-terminal domain of PGIS responds to the facilitation of PGH(2) movement (presentation) from COX to the active site of PGIS, the first 20 residues of PGIS (Delta20-PGIS) were deleted and expressed in COS-7 cells. Delta20-PGIS retained membrane-bound properties and exhibited a slower substrate presentation property. Furthermore, a chimeric molecule (PGIS/TXAS(8-27)) with the replacement of the first 20 residues of PGIS by the corresponding membrane anchor region (residues 8-27) of thromboxane A(2) synthase was created to evaluate the mechanism influencing the biosynthesis of PGI(2) in coordination with COX. The chimera revealed a multiple fold delay in the PGH(2) presentation in low range concentrations of AA (0.3-3muM) at 30s reactions. However, the delay could be recovered by a longer incubation time in high range concentrations of AA (>10muM), but not in low range concentrations of AA. These results demonstrated that the N-terminal domain of PGIS plays a role in the facilitation of the substrate presentation to the PGIS active site in low concentrations of AA, which may be a physiological condition. The TXAS N-terminal domain could not replace the function of the corresponding domain of PGIS, indicating that the facilitation of the substrate presentation is specific.     

On the mechanism of prostacyclin and thromboxane A2 biosynthesis

The present research describes studies which address the mechanism of prostacyclin (PGI2) and thromboxane A2 (TXA2) biosynthesis. In addition to prostaglandin H1 (PGH1), PGG2, PGH2, and PGH3, also 8-iso-PGH2, 13(S)-hydroxy-PGH2, and 15-keto-PGH2 were applied to determine the substrate specificities and kinetics of prostacyclin and thromboxane synthase in more detail. Human platelet thromboxane synthase converted PGH1, 8-iso-PGH2, 13(S)-hydroxy-PGH2 and 15-keto-PGH2 into the corresponding heptadecanoic acid (C17) plus malondialdehyde, whereas the thromboxane derivative was formed only from PGG2, PGH2, and PGH3 together with the corresponding C17 metabolite and malondialdehyde in a 1:1:1 ratio. In contrast, PGG2, PGH2, 13(S)-hydroxy-PGH2, 15-keto-PGH2 and PGH3 were almost completely isomerized to the corresponding prostacyclin derivative by bovine aortic prostacyclin synthase, whereas PGH1 and 8-iso-PGH2 only produced the corresponding C17 hydroxy acid plus malondialdehyde. Isotope-labeling experiments with [5,6,8,9,11,12,14,15-2H]PGH2 revealed complete retention of label and no isotope effect in the course of thromboxane biosynthesis, but the loss of one 2H atom at C-6 with an isotope effect of 1.20 during PGI2 formation. Prostacyclin and thromboxane synthase bind both 9,11-epoxymethano-PGF2 alpha and 11,9-epoxymethano-PGF2 alpha at the heme iron, but according to their difference spectra in opposite ways with respect to the 9- and 11-position. In agreement with published model studies, a cage radical mechanism is proposed for both enzymes according to which the initial radical process is terminated through oxidation of carbon-centered radicals by the iron-sulfur catalytic site, followed by ionic rearrangement to PGI2 or TXA2. Various Fe(III) model compounds as well as liver microsomes or cytochrome P-450CAM can also form small amounts of PGI2 and TXA2, but mainly yield 12(S)-hydroxy-5,8,10-heptadecatrienoic acid plus malondialdehyde probably by a radical fragmentation pathway.