Antithrombotic effect of topically applied 3-Oxa-Methano-prostaglandin I1 in the microcirculation and antiplatelet functions of its active form

The antithrombotic effect of topical application of the 3-oxamethano-prostaglandin (PG) I₁ analog, SM-10902 in the microcirculation and in vitro antiplatelet functions of its active form SM-10906 were estimated in comparison with PGI₂ and PGE₁. In rat platelets, SM-10906 evoked accumulation of intracellular cyclic adenosine 3′,5′-monophosphate, and exhibited antiaggregatory and disaggregatory activities, which were all enhanced by the phosphodiesterase inhibitor theophylline. Additionally, SM-10906 was shown to inhibit platelet adhesion to collagen in human platelet-rich plasma. PGI₂ and PGE₁ also showed in vitro antiplatelet effects in the order of PGI₂ > SM-10906 ≥ PGE₁. SM-10902 exhibited a dose-dependent antithrombotic effect in the guinea pig mesenteric arteriole by a topical application, and this activity might be exerted by the antiplatelet functions of SM-10906. Although SM-10906, PGI₂ and PGE₁ also showed the antithrombotic effects, SM-10902 was the most potent. In conclusion, the present studies indicate that an external topical preparation of SM-10902 may be useful for the therapy of peripheral circulatory insufficiency.     

Prostaglandin E2: Effects on aggregation,shape change and cyclic AMP of rat platelets

PGE₂ inhibits intracellular cyclic AMP accumulation induced by PGE₁ in rat platelets. PGE₂ also counteracts PGE₁-inhibition of both platelet aggregation and shape change. However, in the presence of theophylline, PGE₂ acts like PGE₁ in inhibiting aggregation. The mechanism of interaction of the two closely related prostaglandins is discussed.     

The preparation and characterization of prostaglandin E1 antiserum

Antiserum against PGE₁ was raised in rabbits following immunization with prostaglandin-thyroglobulin conjugates. The antiserum exhibits 22% cross reactivity with PGE₂ but little or no apparent cross reaction with PGE₁ metabolites or heterologous prostaglandins. The data indicate some limitations in the use of this antiserum for the measurement of PGE₁ in biologic samples.     

Inhibition of prostaglandin E2 production by synthetic minor prenylated chalcones and flavonoids: Synthesis,biological activity,crystal structure,and in silico evaluation

The discovery of potent inhibitors of prostaglandin E₂ (PGE₂) synthesis in recent years has been proven to be an important game changer in pharmaceutical industry. It is known that excessive production of PGE₂ triggers a vast array of biological signals and physiological events that contributes to inflammatory diseases such as rheumatoid arthritis, atherosclerosis, cancer, and pain. In this Letter, we report the synthesis of a series of minor prenylated chalcones and flavonoids which was found to be significantly active in suppressing the PGE₂ production secreted by lipopolysaccharide-induced mouse macrophage cells (RAW 264.7). Among the compounds tested, 14b showed a dose-response inhibition of PGE₂ production with an IC₅₀ value of 2.1 μM. The suppression upon PGE₂ secretion was not due to cell death since 14b did not reduce the cell viability in close proximity to the PGE₂ inhibition concentration. The obtained atomic coordinates for the single-crystal XRD of 14b was then applied in the docking simulation to determine the potential important binding interactions with murine COX-2 and mPGES-1 putative binding sites.     

Measurement of prostaglandin E3 and other eicosanoids in biologic samples using high pressure liquid chromatography and radioimmunoassay

A method to measure PGE₃ in biologic samples is described. Complete resolution of PGE₃ from PGE₁ and PGE₂ is achieved by reverse- phase high pressure liquid chromatography. Quantification is carried out by radioimmunoassay using an antibody directed against PGE₂ that has high cross-reactivity with PGE₃. Using this method, a marked increase in PGE₃ production by mouse kidney tissue and in rat urine was demonstrated after supplemental feeding of ω-3 fatty acids. This method can also be applied to measurement of 6-keto-PGF_1α and TXB₂ in the same samples.     

Radioimmunoassay of prostaglandins E1, E2, and F2α in unextracted plasma, serum and myocardium

A radioimmunoassay procedure for the determination of PGE₁, PGE₂, and PGF₂α is presented. The procedure involves the pre-precipitation of each prostaglandin specific antiserum with the precipitating antisera (ARGG), and the use of these antisera mixtures in assaying for PGE₁, PGE₂, and PGF₂α. Applicability of the methods to unextracted plasma, serum and myocardial homogenate has been demonstrated through tests of specificity, recovery, reproducability and parallelism. A mathematical correction for cross-reactivity between PGE₁ and PGE₂, and their opposing antisera is given. To demonstrate the utility of the methodology in differentiation of experimental variables, prostaglandin concentrations in unincubated serum, incubated serum, and the rate of prostaglandin production in serum of dogs are given.     

Prostaglandin E2 production in astrocytes: regulation by cytokines, extracellular ATP, and oxidative agents

Upregulation and activation of phospholipases A₂ (PLA₂) and cyclooxygenases (COX) leading to prostaglandin E₂(PGE₂) production have been implicated in a number of neurodegenerative diseases. In this study, we investigated PGE₂ production in primary rat astrocytes in response to agents that activate PLA₂ including pro-inflammatory cytokines (IL-1β, TNFα and IFNγ), the P2 nucleotide receptor agonist ATP, and oxidants (H₂O₂ and menadione). Exposure of astrocytes to cytokines resulted in a time-dependent increase in PGE₂ production that was marked by increased expression of secretory sPLA₂ and COX-2, but not COX-1 and cytosolic cPLA₂. Although astrocytes responded to ATP or phorbol ester (PMA) with increased cPLA₂ phosphorylation and arachidonic acid release, ATP or PMA only caused a small increase in levels of PGE₂. However, when astrocytes were first treated with cytokines, further exposure to ATP or PMA, but not H₂O₂ or menadione, markedly increased PGE₂ production. These results suggest that ATP release during neuronal excitation or injury can enhance the inflammatory effects of cytokines on PGE₂ production and may contribute to chronic inflammation seen in Alzheimer's disease.     

Interaction between nitric oxide and prostaglandin synthesis in the acute phase of allergic conjunctivitis

Both nitric oxide and prostaglandins induce vasodilatation which is an important feature of local inflammation. The purpose of the study described here was to investigate a possible interaction between these two types of mediators in an experimental model of allergic conjunctivitis. A conjunctival allergic reaction was induced with antigen in sensitized guinea pigs. Conjunctival vascular permeability changes were evaluated with the prophylactic use of an inhibitor of nitric oxide synthase (L-NAME) and a cycloxygenase inhibitor (indomethacin). To study a possible interaction between nitric oxide and prostaglandin synthesis in the acute phase of allergic conjunctivitis, the levels of nitrite and PGE₂ were determined in lavage fluid. The prophylactic use of L-NAME on the formation of conjunctival edema in response to topical PGD₂ administration was studied by measurement of albumin levels in lavage fluid. Both nitric oxide and PGE₂ are synthesized in response to antigen provocation and after histamine administration. Nitric oxide and PGE₂ are produced simultaneously in the conjunctiva and they showed identical synthesis profiles in response to antigen provocation. Pretreatment with L-NAME inhibited the synthesis of PGE₂ whereas exogenous administration of nitric oxide increased the level of PGE₂ in lavage fluid. Prophylactic treatment with L-NAME significantly inhibited the PGD₂ induced albumin extravasation. Nitric oxide seems to play an important role in the acute phase of allergic conjunctivitis it may stimulate PGE₂ production and acts as a secondary mediator in PGD₂ and histamine induced conjunctival edema.     

Rat osteogenic sarcoma cells: Effects of some prostaglandins, their metabolites and analogues on cyclic AMP production

Cyclic AMP production by freshly isolated cells, from a ³²P-induced transplantable rat osteogenic sarcoma, was stimulated by PGE₁, PGE₂ and to a less extent by PGF_2α and PGA₂. In the case of PGE₂, the cyclic AMP content of cells was miximal within 5 min. The 13, 14-dihydro derivatives of PGE₁, PGE₂ and PGF_2α had approximately 40% of the activity of the parent prostaglandin whilst, in every case, the metabolites (15-keto and 13,14-dihydro-15-keto) had very little activity. Two prostaglandin endoperoxide analogues (U44069 and U46619) had only 10% of the activity of an equimolar dose of PGE₂. The data presented in this paper demonstrates similarities between the responses of these cells and cells derived from bony tissue in terms of the ability of prostaglandins to stimulate bone resorption in tissue culture.     

Differential Effects of Prostaglandin E1 and Prostaglandin E2 on Growth and Differentiation of Murine Myeloid Leukemic Cell Line,M1

Effects of prostaglandins (PGs) of the E series on growth and differentiation of murine myeloid leukemic cell line M1 were studied. PGE₁, but not PGE₂, inhibited the growth of M1 cells. PGE₂ neither inhibited nor augmented the antiproliferative effect of PGE₁. PGE₁ augmented the differentiation of M1 cells into macrophage-like cells induced by interleukin 6. PGE₂, however, did not exhibit any effect on the differentiation. PGE₁ caused a marked increase in intracellular cAMP level in M1 cells, whereas PGE₂ had no effect. These results indicate that M1 cells are able to respond only to PGE₁. Radiolabeled PGE₁ binding experiments, however, revealed that there was no specific binding in M1 cells, suggesting that the cells express low numbers of receptors or very low affinity receptors specific for PGE₁. Stable agonists of PGI₂, iloprost, cicaprost or carbacyclin, also potently inhibited the growth of M1 cells. These findings suggest that PGE₁ as well as PGI₂ may play a role in the differentiation of monocyte-macrophage lineage cells.     

A specific radioimmunoassay for PGE2 using an antibody with high specificity and a sephadex LH-20 microcolumn for the separation of prostaglandins

Antiserum against PGE₂ was raised in rabbits following immunization with prostaglandin-hen-γ-globulin conjugate. The antiserum exhibited 14% cross reactivity with PGE₁ and far less cross-reaction with heterologous prostaglandins. A microcolumn of Sephadex LH-20 was used for a partial, but sufficient separation of PGE₂ from PGE₁ and a complete separation from heterologous prostaglandins to ensure a specific RIA for PGE₂. The precision of the method in the range 10–500 picograms showed a coefficient of variation varying between 4 and 13%. The detection limit was 10 picograms corresponding to 15 pg/ml of PGE₂ in serum.In order to demonstrate the validity of the method values obtained for non-diuretic rat renal venous serum were compared with those obtained using the isotope derivative method of Bojesen & Buckhave (1972) on the same samples. The concentrations of PGE₂ obtained were 239 ± 25 pg/ml and 250 ± 58 pg/ml, respectively.     

Prostaglandin E2 modifies SMAD2 and promotes SMAD2–SMAD4 complex formation

We report that PGE₂ promotes Smad2–Smad4 complex formation and this phenomenon could be blocked by DIDS, an anion transporter inhibitor. Our data suggest that PGE₂ had no effects on Smad2 phosphorylation, suggesting that PGE₂-mediated Smad2–Smad4 complex formation is independent of TGF-β signaling and that PGE₂ induced Smad2 modification which is different from TGF-β-mediated phosphorylation. We demonstrate that in primary human glomerular mesangial cells PGE₂ caused modification of Smad2 as detected by Smad2N antibody, raised against a peptide near the N-terminus of Smad2. We hypothesize that Smad2 protein is post-translationaly modified by PGE₂. Direct evidence of Smad2 modification by PGE₂ was achieved by avidin pulldown assay which showed that endogenous Smad2 and recombinant Smad2 protein were attached by biotin-labeled PGE₂. Taken together, our results provided evidence that post-translational modification of Smad2 could be a mechanism for the action of PGE₂ in the pathogenesis of human pathologies.     

Announcement

Antiserum against PGE₁ was raised in rabbits following immunization with prostaglandin-thyroglobulin conjugates. The antiserum exhibits 22% cross reactivity with PGE₂ but little or no apparent cross reaction with PGE₁ metabolites or heterologous prostaglandins. The data indicate some limitations in the use of this antiserum for the measurement of PGE₁ in biologic samples.     

Nasal vasoconstrictor activity of a novel PGE2 analog

A novel PGE₂ analog (CL 116,069) was shown to be effective in dogs as a nasal decongestant. Threshold doses were approximately 0.1 μg/kg with intravenous administration and between 0.08 and 4 μg with topical administration. CL 116,069 was compared to 17-phenyl-trinor PGE₂ (CL 116,147), a compound recently studied in humans, and xylometazoline, a well-known nasal decongestant. When given i.v., efficacious doses of xylometazoline tended to raise blood pressure and be shorter acting than the PGs, which did not affect blood pressure. When given topically, all three were long-acting. CL 116,069 usually had the lowest threshold and CL 116,147 usually induced the smallest response. All three agents were more effective than PGE₁ or PGE₂. A 30-day (b.i.d., topical) toxicity test with CL 116,069 produced no inflammation or nasal pathology and no loss in tissue sensitivity. $\text{In}$$\text{vitro}$ examination of xylometazoline and CL 116,069 for vascoconstrictor activity on dog isolated mucosa revealed a response profile similar to that observed with these agents $\text{in}$$\text{vivo}$; i.e., the magnitude of response was comparable for both agents but the t $\text{1}\text{2}$ was only 74 minutes for xylometazoline and greater that 6.5 hours for CL 116,069. The data suggest that CL 116,069 may provide a therapeutic alternative in which constriction of the nasal blood vessels need not be associated with a generalized vasoconstrictor liability.     

EP2 receptor activation by prostaglandin E2 leads to induction of HO-1 via PKA and PI3K pathways in C6 cells

Recently we proposed that COX-2 induction precedes expression of HO-1 in ischemic preconditioned rat brain. In the current study, we investigated the molecular mechanism by which prostaglandin E₂, one of COX-2 metabolites, induces HO-1 in rat C6 brain cells. We demonstrated that concentration of PGE₂ increased HO-1 expression in C6 cells in vitro. The effects of PGE₂ were mimicked by PGE₂ receptor EP₂ agonists, 11-deoxy PGE₂, and cAMP analog, dibutyl-cAMP. HO-1 expression by PGE₂ was inhibited by LY294002, PI3K inhibitor and H89, PKA inhibitor. The EP₂-specific antagonist, AH8006 also inhibited PGE₂-mediated HO-1 expression in a concentration-dependent manner. Finally, PGE₂ inhibited GOX-induced apoptosis as assayed by FACS analysis or DNA strand breaks assay, and this cell death was reversed by ZnPPIX, HO-1 inhibitor. In addition to HO-1 induction, PGE₂ also increased phosphorylation of Bad by PKA- and PI3K-depednent manner. Taken together, we conclude that PGE₂ induces HO-1 protein expression through PKA and PI3K signaling pathways via EP₂ receptor in C6 cells. The induction of HO-1 along with increase of p-Bad by PGE₂ is responsible for anti-apoptosis against oxidant stress.     

Inhibition of cytotoxic responses by prostaglandin E2 in the presence of interleukin 2

Experiments are described which provide direct evidence for a strong suppressive effect of prostaglandin E₂ (PGE₂) on the activation of cytotoxic T lymphocytes (CTL). The experiments also tested the hypothesis that PGE₂ inhibits cytotoxic responses exclusively by preventing the helper T cells from producing interleukin 2 (IL-2). In support of this hypothesis we found that indomethacin enhanced cytotoxic responses in the absence but not in the presence of concanavalin A-activated spleen cell supernatant (CSCS) indicating that the endogenously produced prostaglandin(s) inhibited primarily the production of IL-2. However, the addition of PGE₂ to microcultures was found to inhibit in a dose-dependent manner the activation of cytotoxic responses against I-region compatible stimulator cells even in the presence of CSCS. This inhibition was not observed in the presence of I-region incompatible stimulator cells, indicating that the inhibitory effect of PGE₂ did not result from a general toxic effect on the culture. The results argue against the notion that PGE₂ inhibits cytotoxic responses exclusively by preventing the production of IL-2.     

Effect of Prostaglandins on Hepatic Cyclic Nucleotide Concentration,Carbohydrate and Lipid Metabolism

The effects of exogenous prostaglandin E₁ (PGE₁) or prostaglandin E₂ (PGE₂) were studied in the isolated perfused rat liver and in the intact canine liver in order to determine the possible physiological role of prostaglandins on hepatic carbohydrate and lipid metabolism. The data indicate that PGE₁ and PGE₂ did not stimulate cyclic AMP (cAMP) and cyclic GMP (cGMP) concentrations in intact dog liver and PGE₁ failed to stimulate cAMP or cGMP in fed or fasted perfused rat liver. PGE₁ did not promote hyperglycemia, glycogenolysis, lipolysis, or prevent epinephrine-induced hyperglycemia in the isolated perfused rat liver. Other known glycogenolytic agents including glucagon and epinephrine increased cAMP and glycogenolysis in the same perfusion system. This study does not support a physiologic role for PGE₁ on hepatic glycogenolysis or lipolysis. If PGE₁ subsequently is found to influence other metabolic parameters such as lipogenesis, gluconeogenesis, ureogenesis or amino acid transport in isolated perfused liver, such alterations would probably occur independent of changes in cyclic nucleotide activity.     

Prostaglandin (PG) accumulation by mouse ovaries, oviducts, and uteri

Uteri, ovaries and oviducts from mice were collected at autopsy. Tissue slices were incubated with [³H]-PGE₂ in the presence or absence of a large excess (100 fold) of nonradioactive PGE₂ using 0.01M sodium phosphate buffer (pH 7.2). Bound and free PGs were separated by a filtration technique. PGE₂ accumulation by the uteri was evaluated as a function of incubation time, wet weight of tissues, and reproductive state. The tissue to medium ratio (T/M) was greater than 1.0 for uteri as the time of incubation increased. This suggests the presence of PGE₂ binding sites in mouse uterine tissue. Also, PGE₂ accumulation was not observed in oviducts or in ovaries.     

Analysis of A,B, E,and F prostaglandins as pentafluorobenzyl esters by electron-capture gas-liquid chromatography

A new and sensitive method is described for the simultaneous analysis of a mixture containing PGE₁, PGE₂, PGF_1α, and PGF_2α by electron-capture gas-liquid chromatography. During derivatization of the mixture, PGE₁ and PGE₂ were converted to PGB₁ and PGB₂, respectively, yielding a mixture of PGB₁, PGB₂, PGF_1α, and PGF_2α trimethylsilyl ether pentafluorobenzyl esters. Gas chromatographic resolution of all four derivatives is sufficient for quantitation of each prostaglandin. The A prostaglandins were analyzed by similar conversion to the respective B prostaglandin derivatives. Minimum detection limits for the B and F prostaglandin derivatives were 10 pg and 1 pg, respectively. Samples of rabbit kidney medulla were incubated and analyzed for A, B, E, and F prostaglandins. The results indicate that the method is capable of high recovery and reproducibility.