Alterations of prostaglandin E2-9-ketoreductase activity in proliferating skin.

The activity of an NADPH-dependent PGE2-9-ketoreductase has been demonstrated in rat and human skin. This activity is localized in the high speed supernatant fraction, indicating the presence of an active PGE2-9-ketoreductase associated with the cytoplasmic fraction of the skin. Transformation of PGE2 into PGF2alpha is enhanced by skin specimens from psoriatic plaques and EFA-deficient rats, both characterized by excessive cellular proliferation and increased NADPH production. Incubations of the 105,000 g supernatant fractions from normal and EFA-deficient rats demonstrated that the activity of the PGE2-9-ketoreductase was elevated in high speed preparations from EFA-deficient rats. Results from these studies suggest that the increased activity of PGE2-9-ketoreductase observed in skin from human psoriatic plaques and EFA-deficient rats may be due in part to the increased generation of NADPH by these tissues and in part to alteration of the PGE2-9-ketoreductase by the excessive proliferation of the tissues.     

Ovarian and uterine prostaglandin E2-9-ketoreductase activity in cyclic and pregnant ewes.

The regulation of luteal function in sheep appears to be dependent in part upon relative utero-ovarian concentrations of PGE2 and PGF2 alpha. Prostaglandin E2-9-ketoreductase converts PGE2 (a putative antiluteolysin) to PGF2 alpha. Enzymatic activity was measured in a cytosolic subcellular fraction of luteal and endometrial tissues collected on days 10, 13 and 16 of the estrous cycle or pregnancy. Respective days represented times before, during, and after the critical period for maternal recognition of pregnancy. Preparations of enzyme were incubated in the presence of tritiated PGE2. Radiolabeled PGF2 alpha (ie., product) was separated from PGE2 by gel filtration chromatography and quantified by liquid scintillation spectrometry. There were no significant differences due to time of tissue collection or pregnancy status in enzymatic activity of luteal tissues. Prostaglandin E2-9-ketoreductase activity isolated from endometria of open ewes was greater than their pregnant counterparts on days 13 and 16. Thus, the potential capacity of the ovine uterus to generate luteolytic PGF2 alpha from PGE2 substrate is elevated during an infertile estrous cycle.     

Selective stimulation of venous prostaglandin E 9-ketoreductase by bradykinin

The effects of bradykin on prostaglandin metabolism in canine mesenteric vessels were examined. Bradykinin stimulated microsomal prostaglandin synthesis in both artery and vein; this stimulation was more pronounced when [¹⁴C]phosphatidylcholine rather than [¹⁴C]arachidonate was used as the substrate for prostaglandin synthetase. This suggested that bradykinin enhanced a membrane phospholipase. In addition, bradykinin selectively stimulated prostaglandin E 9-ketoreductase activity from veins but not arteries. This may explain the finding that bradykinin induces the release of prostaglandin E compounds from arteries but prostaglandin F compounds from veins.     

Demonstration of a thyrotropin-responsive prostaglandin E2-9-ketoreductase in bovine thyroid.

1. Prostaglandin E2-9-ketoreductase activity was demonstrated in bovine thyroid homogenates. 2. The enzyme requires reduced pyridine nucleotide and dialysis prior to assay for optimal activity. 3. The products of the reaction, NADP and prostaglandin F2alpha, inhibit enzyme activity. 4. Sigmoidal kinetics are observed when substrate concentration is plotted against enzyme velocity, indicative of an allosteric enzyme. 5. Thyrotropin increases enzyme activity in bovine thyroid slices. This increase is both hormone- and tissue-specific.     

Purification and regulatory properties of chicken heart prostaglandin E 9-ketoreductase.

Prostaglandin E 9-ketoreductase was purified from chicken heart by ammonium sulfate fractionation, and DEAE-Sephadex, hydroxylapatite and phosphocellulose chromatography. Two peaks of activity were resolved during the phosphocellulose chromatographic step. Both peaks were stimulated by a substance that was not bound to the phosphocellulose column. This stimulatory substance was destroyed by treatment with phosphodiesterase and 0.1 M NaOH. It was heat-stable (100 degrees, 2 min), nondialyzable, and resistant to treatment with pronase, ribonuclease, and deoxyribonuclease; but it was dialyzable after heating or digestion with pronase. Sodium pyrophosphate also enhanced the activities of the prostaglandin E 9-ketoreductases as did angiotensin I; but not angiotensin II. In the presence of 3':5'-cyclic AMP, AMP, or several other ribonucleotides, the enhancing effects of the natural stimulatory substance, sodium pyrophosphate or angiotensin I were blocked, but these ribonucleotides themselves had little effect on the enzymes activity. The substrate specificities of the two prostaglandin E 9-ketoreductases were also studied. Both the 9-keto group and the 15-keto group of 15-ketoprostaglandin F2 alpha could be converted to the corresponding hydroxyl group; the 15-keto group was reduced faster than the 9-keto group. Prostaglandin D2, a prostaglandin with a 9-hydroxyl and an 11-keto group, could not be converted to prostaglandin F2 alpha nor could cyclohexanone be converted to cyclohexanol by the prostaglandin E 9-ketoreductase.     

Nitric oxide production in murine spleen cells: role of interferons and prostaglandin E(2) in the generation of cytotoxic activity

The production of nitric oxide (NO) was measured in cultures of spleen cells stimulated by lipopolysaccharide (LPS), IL-2 or LPS + IL-2. We observed that NO synthesis is increased by IFN-gamma but inhibited by IFN-alpha/beta. This is not the case when IL-2 is present in the cultures, since interferons play a minor role in the regulation of the NO production. When IL-2 and LPS were associated in the cultures, the IFN-alpha/beta role seems more important than that of IFN-gamma. PGE(2) inhibits NO production in LPS supplemented cultures but has a slight effect in the presence of IL-2 and no effect with IL-2 + LPS. 3-isoButyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases, induces a decrease of IFN production. In the presence of H-7, an inhibitor of protein kinase C (PKC), NO production is reduced when the cultures are supplemented by LPS or IL-2 but not when IL-2 and LPS are both added. H-7 also reduced IFN production. In the presence of N(G)-monomethyl-L-arginine (N-MMA), an inhibitor of NO synthesis, IFN production was increased, with no change in the cytotoxic activity. Hence, interferons regulate NO production by mouse spleen cells and, in return, NO modulates the generation of IFN.     

Prostaglandin E2-9-ketoreductase of rat testis.

A divalent metal dependent gluconolactonase has been isolated from porcine liver and purified to apparent homogeneity. Its molecular weight is estimated at 223,000 and that of the subunits is 37,200 as determined by gel electrophoresis. A K_m value of 6.2 mM was obtained at 27° in 50 mM tris HCl buffer. Gluconolactonase is specific for gluconolactone, and manganese is preferred over magnesium for maximum activity. The hepatic concentration of gluconolactonase is estimated to be 7.2 μmol of enzyme per kg of porcine liver, and a subcellular fractionation study indicates that this enzyme is located primarily within the cytosol.     

Metabolism of prostaglandin E1 and of glutathione conjugate of prostaglandin A1 (GSH-prostaglandin A1) by prostaglandin 9-ketoreductase from rabbit kidney.

Rabbit kidney prostaglandin 9-ketoreductase was found to metabolize the glutathione conjugate of prostaglandin A1 (GSH-prostaglandin A1). Apparent Km (GSH-prostaglandin A1) 13 microM and apparent Km (prostaglandin E1) 200 microM. The cytosolic preparation was subjected to gelfiltration and isoelectric focusing, which revealed that metabolism of prostaglandin E1 and GSH-prostaglandin A1 occurs by means of the same fractions. Furthermore, prostaglandin E1 and GSH-prostaglandin A1 are competitive inhibitors of the enzyme, when GSH-prostaglandin A1 and prostaglandin E1 are tested as substrates, respectively. It si concluded, that GSH-prostaglandin A1 is a much better substrate for prostaglandin 9-ketoreductase from rabbit kidney than is prostaglandin E1.     

Prostaglandin-E2 9-ketoreductase from human uterine decidua vera

Prostaglandin-E2 9-ketoreductase, the enzyme which catalyzes the reaction from prostaglandin E2 (PGE2) to prostaglandin F2 alpha (PGF2 alpha), has been purified 232-fold from human uterine decidua vera. The molecular mass of the enzyme, as estimated by fast protein liquid chromatography, was 29 kDa. Sodium dodecyl sulfate disc gel electrophoresis of the denatured enzyme revealed a molecular mass of 31 kDa. These data suggest that the enzyme consists of a single polypeptide chain. The rate equation of the enzyme reaction for two substrates was used for the determination of five kinetic constants. The equilibrium constant with respect to PGE2 was 83 microM, the Michaelis constant, Km, for PGE2 was 93 microM. For NADPH, the equilibrium constant was 1.0 microM and Km was 1.6 microM. The maximal velocity for the forward reaction was V1 = 217 pmol/min. The inhibition constants for the analgesic agents indomethacin and fentiazac were Ki = 850 microM and Ki = 450 microM and for the steroid progesterone Ki = 1.5 mM, respectively. Prostaglandin-E2 9-ketoreductase might be responsible for the control of the PGE2/PGF2 alpha ratio in human decidua vera. The enzyme, therefore, might be an important factor in the cascade of events leading to uterine contractions and parturition.     

Partial purification of prostaglandin E2-9-ketoreductase and prostaglandin-15-hydroxydehydrogenase from ovarian tissues of rabbits

Prostaglandin E2-9-ketoreductase (PGE2-9-KR) and prostaglandin-15-hydroxydehydrogenase (PG-15-HDH) have been purified 25.0- and 15.4-fold, respectively. The rate equations of the enzyme reaction for two substrates were used for the determination of kinetic constants. The Michaelis constant, Km, for PGE2 was 122 microM for the PGE2-9-KR and 8 microM for the PG-15-HDH. The presence of both enzymes in ovarian tissues of rabbits indicate that these tissues may be able to synthesize and metabolize PGF2 alpha.     

Evidence for a role of phosphodiesterase 4 in lipopolysaccharide-stimulated prostaglandin E2 production and matrix metalloproteinase-9 activity in human amniochorionic membranes

Chorioamniotic infection is a leading cause of preterm premature rupture of fetal membranes (amnion and chorion). Bacterial infection induces an inflammatory response characterized by elevated production of proinflammatory cytokines; the latter activate the production of both PGs that stimulate uterine contractions, and matrix metalloproteinases (MMPs) that degrade the extracellular matrix of the chorioamniotic membranes. The inflammatory response is under the control of cAMP content, which is partly regulated by phosphodiesterases (PDE). In this study, we investigated the role of the PDE4 family in the inflammatory process triggered by LPS in a model of amniochorionic explants. We found that PDE4 family is the major cAMP-PDE expressed in human fetal membranes and that PDE4 activity is increased by LPS treatment. Selective inhibition of PDE4 activity affected LPS signaling, because PDE4 inhibitors (rolipram and/or cilomilast) reduced the release of the proinflammatory cytokine TNF-alpha and increased the release of the anti-inflammatory cytokine IL-10. PDE4 inhibition reduced cyclooxygenase-2 protein expression and PGE(2) production and also modulated MMP-9, a key mediator of the membrane rupture process, by inhibiting pro-MMP-9 mRNA expression and pro-MMP-9 activity. These results demonstrate that the PDE4 family participates in the regulation of the inflammatory response associated with fetal membrane rupture during infection. The PDE4 family may be an appropriate pharmacological target for the management of infection-induced preterm delivery.     

Effects of gonadectomy and steroid treatment on renal prostaglandin 9-ketoreductase activity in the rat

The effect of gonadectomy and treatment with sex-steroids on renal prostaglandin 9-ketoreductase activity in 10-11 week old male and female rats was determined. Rats were gonadectomized or subjected to sham operation at 3 weeks of age. During week 7, rats were injected s.c. twice over a 6-day interval with vehicle (peanut oil, 0.5 ml X kg-1) or with depot forms of testosterone (5 mg X kg-1), estradiol (0.02 mg X kg-1), progesterone (5 mg X kg-1), or estradiol and progesterone combined. Renal prostaglandin 9-ketoreductase activity was about 50% higher in female rats than in males. Gonadectomy decreased 9-ketoreductase activity in females, but not in males, and eliminated the gender difference in enzyme activity. Treatment with estradiol elevated 9-ketoreductase activity in males and females, while treatment with testosterone or progesterone was without effect. Progesterone did, however, antagonize the elevation in 9-ketoreductase activity produced by estradiol.     

Nitric oxide inhibits cell growth in cultured human thyrocytes

Effect of NO induced by interleukin-1 (IL-1) or IL-1/interferon-_γ (IL-1/IFN-_γ) was investigated on cell growth using primary cultures of human thyrocytes. Cytokine-induced NO production was associated not only with an increase in cyclic GMP (cGMP) formation but also with an inhibition of cell growth determined by bromo-deoxyuridine (Br-dU) incorporation into DNA. When NO synthesis was blocked by N^G-monomethyl-L-arginine (L-MMA), cGMP formation was prevented in parallel with NO production and inversely a restoration of cell growth was evident. S-nitroso-N-acetyl-penicillamine, a NO donor, but not a cell permeable cGMP analog, 8-bromo-cGMP, inhibited cell growth in a dose-dependent manner. The present findings strongly indicate that endogenous NO produced by the cytokine treatment as well as exogenous NO, has a cGMP-independent inhibitory action on human thyrocyte growth.     

Nitric oxide inhibits thrombin receptor-activating peptide-induced phosphoinositide 3-kinase activity in human platelets.

Although nitric oxide (NO) has potent antiplatelet actions, the signaling pathways affected by NO in the platelet are poorly understood. Since NO can induce platelet disaggregation and phosphoinositide 3-kinase (PI3-kinase) activation renders aggregation irreversible, we tested the hypothesis that NO exerts its antiplatelet effects at least in part by inhibiting PI3-kinase. The results demonstrate that the NO donor S-nitrosoglutathione (S-NO-glutathione) inhibits the stimulation of PI3-kinase associated with tyrosine-phosphorylated proteins and of p85/PI3-kinase associated with the SRC family kinase member LYN following the exposure of platelets to thrombin receptor-activating peptide. The activation of LYN-associated PI3-kinase was unrelated to changes in the amount of PI3-kinase physically associated with LYN signaling complexes but did require the activation of LYN and other tyrosine kinases. The cyclic GMP-dependent kinase activator 8-bromo-cyclic GMP had similar effects on PI3-kinase activity, consistent with a model in which the cyclic nucleotide mediates the effects of NO. Additional studies showed that wortmannin and S-NO-glutathione have additive inhibitory effects on thrombin receptor-activating peptide-induced platelet aggregation and the surface expression of platelet activation markers. These data provide evidence of a distinct and novel mechanism for the inhibitory effects of NO on platelet function.     

Nitric oxide (NO)-related pharmaceuticals: contemporary approaches to therapeutic NO modulation

The biologically important gaseous radical, nitric oxide (NO), is a versatile chemical entity that enters into regulatory, protective, and adverse interactions with biomolecules and cells, in some cases through NO-derived nitrogen oxide species. Both excess tissue NO and its insufficiency have been implicated in the genesis or evolution of several important disease states. The associated medical needs and commercial opportunities have fostered attempts to modulate tissue NO tone for symptomatic benefit or therapeutic gain. State-of-the-art strategies for NO modulation in contemporary drug discovery and development encompass sexual dysfunction, cardiovascular, and antiinflammatory indications. Increased understanding of NO's physiological chemistry and ways to target its pharmacology appear critical to the successful clinical exploitation of NO's diverse properties. Integration of research on both the basic science of NO's mechanistic biology and the applied science of drug discovery and development represents a millennium mandate to the pharmaceutical industry in the area of NO-related therapeutics.     

Nitric oxide (NO) synthase immunoreactivity in the starfish Marthasterias glacialis

The neuroendocrine system of the starfish Marthasterias glacialis was investigated immunocytochemically using antisera specific for rat neuronal, bovine aortic endothelial, and mouse macrophage, nitric oxide (NO) synthases. Immunoreactivity was detected only with the antibodies specific for the neural enzyme, in the ectoneural and hyponeural tissues of the radial nerve cords and in the basiepithelial plexus and endocrine cells of the digestive tract. The pyloric stomach showed more immunoreactive structures than the other digestive organs, with the rectal caeca showing the least activity. Immunoreactive endocrine cells were located in the cardiac and pyloric stomachs and in the pyloric caeca. Co-localization of the enzyme immunoreactivity, and the staining for NADPH-diaphorase, demonstrate the presence of NO synthase in echinoderms. These results provide further evidence that NO is a neuronal messenger of early phylogenetic origin which has been conserved throughout evolution.     

Nitric oxide regulates adenylyl cyclase activity in rat striatal membranes

The regulation of adenylyl cyclase activity by nitric oxide (NO) was studied in rat (Sprague-Dawley) striatal membranes. Three chemically distinct NO donors attenuated forskolin-stimulated activity but did not alter basal activity. Maximum inhibition resulted in a 50% decrease in forskolin-stimulated activity, consistent with the presence of multiple isoforms of adenylyl cyclase and our previous findings that only the forskolin-stimulated activity of the type-5 and -6 isoform family of enzymes is inhibited by NO. To monitor primarily the type-5 isoform, we examined the ability of NO donors to attenuate D(1)-agonist-stimulated adenylyl cyclase activity. Under those conditions, complete inhibition was observed. The data indicate that NO attenuates neuromodulator-stimulated cAMP signaling in the striatum.     

Exogenous prostaglandin E2 inhibits TPA induced matrix metalloproteinase-9 production in MCF-7 cells

Elevated levels of prostaglandin E2 (PGE2) have been reported in many high metastatic human breast cancers, but no relationship between exogenous PGE2 activity, expression of matrix metalloproteinases (MMPs) and metastasis in human tumor cells has been reported. The poorly invasive human breast cancer cell line MCF-7 was cultured for 24h in the presence of both phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 nM) and PGE2 (1 microM) and the activity of MMP-9, one of the MMPs involved in metastasis, was measured, in growth medium by gelatin substrate zymography. TPA induced a strong production of MMP-9 while exogenous PGE2 had no effect on the basal MMP-9 level, but inhibited the TPA induced enzyme expression and matrigel invasiveness. We showed that MCF-7 cells expressed EP2, EP3 and EP4 receptors for PGE2 and that its action was probably mediated by EP4 receptor and adenylyl cyclase activation while cAMP dependent PKA was not involved in the process of inhibition of MMP-9 production. These findings suggest a possible inhibitory role for exogenous PGE2 in the metastatic process development.