Prostaglandin E<Subscript>2</Subscript> (PGE<Subscript>2</Subscript>) suppresses natural killer cell function primarily through the PGE<Subscript>2</Subscript> receptor EP4

The COX-2 product prostaglandin E₂ (PGE₂) contributes to the high metastatic capacity of breast tumors. Our published data indicate that inhibiting either PGE₂ production or PGE₂-mediated signaling through the PGE₂ receptor EP4 reduces metastasis by a mechanism that requires natural killer (NK) cells. It is known that NK cell function is compromised by PGE₂, but very little is known about the mechanism by which PGE₂ affects NK effector activity. We now report the direct effects of PGE₂ on the NK cell. Endogenous murine splenic NK cells express all four PGE₂ receptors (EP1-4). We examined the role of EP receptors in three NK cell functions: migration, cytotoxicity, and cytokine release. Like PGE₂, the EP4 agonist PGE₁-OH blocked NK cell migration to FBS and to four chemokines (ITAC, MIP-1α, SDF-1α, and CCL21). The EP2 agonist, Butaprost, inhibited migration to specific chemokines but not in response to FBS. In contrast to the inhibitory actions of PGE₂, the EP1/EP3 agonist Sulprostone increased migration. Unlike the opposing effects of EP4 vs. EP1/EP3 on migration, agonists of each EP receptor were uniformly inhibiting to NK-mediated cytotoxicity. The EP4 agonist, PGE₁-OH, inhibited IFNγ production from NK cells. Agonists for EP1, EP2, and EP3 were not as effective at inhibiting IFNγ. Agonists of EP1, EP2, and EP4 all inhibited TNFα; EP4 agonists were the most potent. Thus, the EP4 receptor consistently contributed to loss of function. These results, taken together, support a mechanism whereby inhibiting PGE₂ production or preventing signaling through the EP4 receptor may prevent suppression of NK functions that are critical to the control of breast cancer metastasis.     

Relative contracting adn relaxing potencies of a series of prostaglandins on isolated canine mesenteric artery strips

The contracting and relaxing potencies of anf interactions between a number of prostaglandins (PGs) were studied $\text{in vitro}$ on spiral strips of small canine mesenteric arteries (outside diameter < mm). PGF₂α and PGE₂, the most potent contracting PGs, were nearly equal in potency (EC₅₀ 4 × 10^?7M) and did not cause relaxation under our experimental conditions. PGI₂ and PGE₁ were equal and the most potent relaxing PGs (EC₅₀ 3 × 10^?9M). PGE₁ also caused contraction, but this effect was not consistent. PGI₂ did not cause contraction in concentrations up to 3 × 10^?6M. In higher concentrations, however, it caused abrupt and near maximal contraction. PGD₂ was weak in both respect, causing incomplete relaxation and contraction or biphasic effects. Interaction studies showed that PGE₁ and PGI₂ mutually excluded the relaxing effects of each other. PGE₁ also reversed the relaxing effect of isoproterenol. However, pre-exposure to PGD₂ did not attenuate the relaxing effect of PGE₁ or PGI₂ nor was the relaxing effect of PGD₂ changed by pre-exposure to PGE₁. Two different orders of potency of PGs suggest two PG receptors subserving contraction and relaxation, respectively. Further, it appears that several PGs can act upon both receptors which may explain unusual interactions between the PGs and some of their atypical effects. Finally, the data also suggest that there may be subtypes of the PG receptors subserving contraction and relaxation.     

Maturation of the contractile response of the Emu ductus arteriosus

The avian embryo has a pair of ductus arteriosi that allow the blood to bypass the pulmonary circulation prior to the initiation of lung ventilation. Our objective was to characterize the factors regulating DA tone during the later stages of development in the emu embryo. We examined in vitro the reactivity of the emu ductus from day 39 through 49 of a 50-day incubation. Steady state tension was not altered by the COX inhibitor indomethacin or the nitric oxide synthase inhibitor l-NAME. However, prostaglandin E₂ (PGE₂) produced a significant relaxation. Norephinephrine and U-46619 produced strong significant contractions in the emu DA and the adrenergic response matured with development. The contractile response to oxygen matured as the embryo developed with significant oxygen-induced contraction on days 45 and 49, but not on day 39 of incubation. The Kv channel inhibitor 4-aminopyridine induced the contraction of the day 48–49 ductus of similar magnitude as the oxygen-induced contraction. The oxygen-induced contraction was reversed by the reducing agent DTT and the electron transport chain inhibitor rotenone. These results suggest that while the emu DA responds to PGE₂, locally produced PGE₂ are not the important regulators of vessel tone. Additionally, relaxation upon addition of the mitochondria electron transport chain inhibitor rotenone suggests that the mitochondria might be acting as vascular oxygen sensors in this system through the production of reactive oxygen species to stimulate the oxygen-induced contraction in a similar fashion to mammals.     

Specific receptors for prostaglandins in airways

The relative bronchomotor activities of prostaglandins (PG) E₁, E₂, F_2α, F_2β and I₂ and of three synthetic E prostaglandin analogues (TR4161, TR4367 and TR4752) were determined on a large number of isolated preparations of guinea-pig trachea and human bronchial muscle. Each prostaglandin was capable of eliciting both contraction and relaxation, the relative incidence of these responses partly depending on concentration. TR4161 was a virtually pure relaxant; TR4367 was virtually devoid of bronchomotor activity; and TR4752 was a potent relaxant, devoid of contractant activity. The results also provided distinct rank orders of approximate potency for contraction and relaxation. Tachyphylaxis to the relaxant activities of PGE₁ and TR4752 confirmed the underlying contractant activity of the two natural E prostaglandins. Antagonism with a high dose of indomethacin of the contractant actions of PGE₁, PGE₂ and PGF_2α confirmed the presence of relaxant activities in each.Inhaled aerosols of the same natural and synthetic prostaglandins were evaluated for irritant activity on the airways, using the cough response of the restrained conscious cat. All of them, except TR4161, elicited severe coughing. The rank order of potencies for irritancy differed from those for tracheobronchial contractant and relaxant activities.These findings suggest that the three responses studied arise from the activation of three distinct PG receptors in the airways. We propose the terms χ (contractant), ψ (relaxant) and ω (irritant) for these putative receptors for prostaglandins or possibly other prostanoids.     

Effects of increasing concentrations of sodium metabisulphite (Na<Subscript>2</Subscript>S<Subscript>2</Subscript>O<Subscript>5</Subscript>, SBS) on deoxynivalenol (DON) concentration and microbial spoilage of triticale kernels preserved without and with propionic acid at various moisture contents

Unground triticale kernels contaminated with 6.63 mg deoxynivalenol (DON) per kg dry matter were stored for up to 63 days at total moisture contents of 13 and 15% in order to study the time-dependent kinetics of DON concentration in dependence on graded levels of sodium metabisulfite [0, 1, 2, 3, 4 and 5 g Na₂S₂O₅ (SBS) per kg], and in the absence and presence of 10 g propionic acid (PA) per kg. The DON concentration decreased with increasing amounts of supplemented SBS and with increasing duration of the preservation period in a bi-exponential fashion when SBS addition was ≥3 g/kg. Lower SBS concentrations yielded inconsistent results. The maximum measured DON reductions after adding 5 g SBS/kg were 3 and 4% of the initial DON concentration after 63 days in the absence and presence of PA at moisture contents of 15%, while the corresponding recovery for the variants preserved at 13% amounted to 21 and 11%, respectively. The 12 variants preserved without PA supplementation were more frequently contaminated by moulds and yeasts (n = 5) than the corresponding variants stored together with PA (n = 1). The overall results and regressive evaluations do suggest that the highest SBS addition of 5 g/kg triticale at a moisture content of 15% preserved for 63 days would be necessary for a maximum DON reduction. Although PA did not exert a direct decontaminating effect, an additional supplementation together with SBS seemed to be advantageous with regard to the prevention of yeast and mould contamination and favouring the decontamination reaction by the acid milieu.     

Prostaglandin I2 is less relaxant than prostaglandin E2 on the lamb ductus arteriosus

Prostaglandin(PG) I₂ and its stable metabolite, 6-keto-PGF_1α, were tested on the isolated ductus arteriosus from mature fetal lambs. PGI₂ relaxed the ductus in high doses (threshold 10⁻⁶M) and its activity disappeared on standing at room temperature for 30 minutes. 6-keto-PGF_1α was inactive at all doses. By contrast, PGE₂ produced a dose-dependent relaxation over a range between 10⁻¹⁰ and 10⁻⁶ M. These findings confirm that PGE₂ is the most potent ductal relaxant among the known derivatives of arachidonic acid. PGE₂ probably maintains ductus patency in the fetus and, together with PGE₁, remains the compound of choice in the management of newborns requiring a viable ductus for survival.     

Effects of prostaglandin E1, I2 and isoproterenol on the tissue cyclic AMP content in longitudinal muscle of rabbit intestine

Effects of prostaglandin E₁(PGE₁) and prostaglandin I₂(PGI₂) on the mechanical activity and tissue cyclic AMP content of the longitudinal muscle of rabbit intestine were examined, comparing that of isoproterenol. PGE₁ or PGI₂ caused a contraction and did not affect the tissue cyclic AMP content. Isoproterenol caused a relaxation and increasedtissue cyclic AMP content.     

Transient-state biodegradation behavior of a horizontal biotrickling filter in co-treating gaseous H<Subscript>2</Subscript>S and NH<Subscript>3</Subscript>

A horizontal biotrickling filter (HBTF) was used to inoculate autotrophic sulfide-oxidizing and ammonia-oxidizing microbial consortiums over H₂S-exhausted carbon for co-treating H₂S and NH₃ waste gas in a long-term operation. In this study, several aspects (i.e., pH change, shock loading and starvation) of the dynamic behavior of the HBTF were investigated. The metabolic products of N and S bearing species in recycling liquid and biological activities of the biofilm were analyzed to explain the observed phenomena and further explore the fundamentals behind. In the pH range of 4–8.5, although the removal efficiencies of H₂S and NH₃ remained 96–98% and 100%, respectively, the metabolic products demonstrated different removal mechanisms and pathways. NH₄-N and NO₂/NO₃-N were dominated at pH ≤6 and ≥7, respectively, indicating the differentiated contributions from physical/chemical adsorption and bio-oxidation. Moreover, the HBTF demonstrated a good dynamic stability to withstand shock loadings by recovering immediately to the original. During shock loading, only 15.4% and 17.9% of captured H₂S and NH₃ was biodegraded, respectively. After 2, 11, and 48 days of starvation, the HBTF system reached a full performance within reasonable re-startup times (2–80 h), possibly due to the consumption of reduced S and N species in biomass or activated carbon thus converted into SO₄-S and NO₃-N during starvation period. The results helped to understand the fundamental knowledge by revealing the effects of pH and transient loadings linked with individual removal mechanism for H₂S and NH₃ co-treatment in different conditions.     

Differential release of prostaglandins by organ cultures of human fetal trachea and lung

Summary Human fetal lung at 16–19 weeks gestation has a partially differentiated epithelium, and in organ culture, distal airsacs dilate and the epithelium autodifferentiates to type I and II pneumatocytes, processes regulated by endogenous prostaglandin PGE₂. Human fetal trachea, at the same gestation, has a terminally differentiated mucociliary epithelium but after 4–6 d in organ culture, develops squamous metaplasia. Tracheal cultures restricted to 3 d have normal phase-contrast and light microscopy appearances and immunohistochemical reactivities (epithelium: cytokeratin 7,8,18; glutathione S-transferase pi-isozyme; epithelial membrane antigen and mesenchyme; desmin; vimentin). In human fetal trachea organ cultures, the predominant prostaglandins released are 6-keto-PGF_1α, PGF_2α, and PGE₂, a pattern similar to that previously described for human adult trachea and lung. In fetal lung cultures, 13,14-dihydro-15-keto-PGF_2α is the major prostaglandin released with lesser amounts of 13,14-dihydro-15-keto-PFG_2α, PGF_2α, PGE₂, and 6-keto-PGF_1α. Human fetal lungin vitro has the competence to self-differentiate, as early as 12 weeks gestation and presence of high levels in fetal lung of the inactive metabolite 13,14-dihydro-15-keto-PGE₂ relative to PGE₂ suggests that active prostaglandin catabolism may be one of the mechanisms to retard this stage of maturationin vivo by limiting PGE₂ availability. Surprisingly, the profile of prostaglandins released from fetal lung organ culture does not change to that of a mature lung with terminal differentiation of the epithelium, and this may indicate differences in the expression of key prostaglandin-metabolizing enzymes in developing human fetal lung in culture and within utero ontogeny.     

Effects of temperature,photosynthetic photon flux density,photoperiod and O<Subscript>2</Subscript> and CO<Subscript>2</Subscript> concentrations on growth rates of the symbiotic dinoflagellate, <Emphasis Type="Italic">Amphidinium</Emphasis> sp.

Symbiotic dinoflagellates of the species Amphidinium are expected to be pharmaceutically useful microalgae because they produce antitumor macrolides. A microalgae production system with a large number of cells at a high density has been developed for the efficient production of macrolide compounds. In the present study, the effects of culture conditions on the cellular growth rate of dinoflagellates were investigated to determine the optimum culture conditions for obtaining high yields of microalgae. Amphidinium species was cultured under conditions with six temperature levels (21–35°C), six levels of photosynthetic photon flux density (15–70 μmol photons m⁻² s⁻¹), three levels of CO₂ concentration (0.02–0.1%), and three levels of O₂ concentration (0.2–21%). The number of cells cultured in a certain volume of solution was monitored microscopically and the cellular growth rate was expressed as the specific growth rate. The maximum specific growth rate was 0.022 h⁻¹ at a temperature of 26°C and O₂ concentration of 5%, and the specific growth rate was saturated at a CO₂ concentration of 0.05%, a photosynthetic photon flux density of 35 μmol photons m⁻² s⁻¹ and a photoperiod of 12 h day⁻¹ upon increasing each environmental parameter. The results demonstrate that Amphidinium species can multiply efficiently under conditions of relatively low light intensity and low O₂ concentration.     

Subsurface CO<Subscript>2</Subscript> Dynamics in Temperate Beech and Spruce Forest Stands

Rates of soil respiration (CO₂ effluxes), subsurface pore gas CO₂/O₂ concentrations, soil temperature and soil water content were measured for 15 months in two temperate and contrasting Danish forest ecosystems: beech (Fagus sylvatica L.) and Norway spruce (Picea abies [L.] Karst.). Soil CO₂ effluxes showed a distinct seasonal trend in the range of 0.48–3.3 μmol CO₂ m⁻² s⁻¹ for beech and 0.50–2.92 μmol CO₂ m⁻² s⁻¹ for spruce and were well-correlated with near-surface soil temperatures. The soil organic C-stock (upper 1 m including the O-horizon) was higher in the spruce stand (184±23 Mg C ha⁻¹) compared to the beech stand (93±19 Mg C ha⁻¹) and resulted in a faster turnover time as calculated by mass/flux in soil beneath the beech stand (28 years) compared to spruce stand (60 years). Observed soil CO₂ concentrations and effluxes were simulated using a Fickian diffusion-reaction model based on vertical CO₂ production rates and soil diffusivity. Temporal trends were simulated on the basis of observed trends in the distribution of soil water, temperature, and live roots as well as temperature and water content sensitivity functions. These functions were established based on controlled laboratory incubation experiments. The model was successfully validated against observed soil CO₂ effluxes and concentrations and revealed that temporal trends generally could be linked to variations in subsurface CO₂ production rates and diffusion over time and with depths. However, periods with exceptionally high CO₂ effluxes (> 20 μmol CO₂ m⁻² s⁻¹) were noted in March 2000 in relation to drying after heavy rain and after the removal of snow from collars. Both cases were considered non-steady state and could not be simulated.     

Differences between cellular mechanisms of ATP-and noradrenaline-induced inhibition of visceral smooth muscles under conditions of selective and combined activation of M<Subscript>2</Subscript> or M<Subscript>3</Subscript> cholinoreceptors

Our studies of the role of phospholipase C in inhibitory synaptic action upon visceral smooth muscles demonstrated that, under conditions of carbachol (CCh)-induced pre-activation of cholinoreceptors, ATP-or noradrenaline (NA)-evoked relaxation of these muscles is mediated by the phospholipase C-independent pathway, while the phospholipase C-dependent pathway does not manifest itself as a mechanism that determines the inhibitory effect of the above transmitters. Under conditions of pre-activation of muscarinic cholinoreceptors, ATP-and NA-induced relaxation is continued due to activation of inositol trisphosphate (InsP₃)-sensitive receptors despite the fact that the pathway of inhibition is phospholipase C-independent. This is confirmed by complete depression of the inhibitory effects of ATP and NA against the background of CCh-induced contraction after pre-incubation of the studied preparations together with 100 μM 2-APB, a blocker of InsP₃ receptors. Selective blockings of either M₂ or M₃ cholinoreceptors are accompanied by a complete loss of the ability of the above blocker of InsP₃ receptors (2-APB) to suppress ATP-and NA-induced contraction of smooth muscles in the state of CCh-induced contraction. It can be hypothesized that, under conditions of selective pre-activation of M₂ or M₃ cholinoreceptors, the mechanisms of intracellular signalling mediating the inhibition events are modified. The InsP₃-dependent pathway that determines both adrenergic and purinergic inhibition of smooth muscles is switched off, and the inhibitory action of neurotransmitters is realized under such conditions through the InsP₃-independent pathway. Therefore, in our study we first found differences between cellular mechanisms underlying ATP-and NA-induced inhibition of smooth muscles under conditions of selective activation of either M₂ or M₃ cholinoreceptors and the mechanisms underlying the relaxing action of inhibitory neurotransmitters under conditions of combined synergistic activation of cholinoreceptors of both the above-mentioned subtypes. Neirofiziologiya/Neurophysiology, Vol. 39, No. 1, pp. 22–31, January–February, 2007.     

Molecular modeling and biophysical analysis of the c-MYC NHE-III<Subscript>1</Subscript> silencer element

G-Quadruplex and i-Motif-forming sequences in the promoter regions of several oncogenes show promise as targets for the regulation of oncogenes. In this study, molecular models were created for the c-MYC NHE-III₁ (nuclease hypersensitivity element III₁) from two 39-base complementary sequences. The NHE modeled here consists of single folded conformers of the polypurine intramolecular G-Quadruplex and the polypyrimidine intramolecular i-Motif structures, flanked by short duplex DNA sequences. The G-Quadruplex was based on published NMR structural data for the c-MYC 1:2:1 loop isomer. The i-Motif structure is theoretical (with five cytosine–cytosine pairs), where the central intercalated cytosine core interactions are based on NMR structural data obtained for a tetramolecular [d(A₂C₄)₄] model i-Motif. The loop structures are in silico predictions of the c-MYC i-motif loops. The porphyrin meso-tetra(N-methyl-4-pyridyl)porphine (TMPyP4), as well as the ortho and meta analogs TMPyP2 and TMPyP3, were docked to six different locations in the complete c-MYC NHE. Comparisons are made for drug binding to the NHE and the isolated G-Quadruplex and i-Motif structures. NHE models both with and without bound cationic porphyrin were simulated for 100 ps using molecular dynamics techniques, and the non-bonded interaction energies between the DNA and porphyrins calculated for all of the docking interactions. Figure Molecular models of the average structure of the final 20 ps of the molecular dynamics simulation of the c-MYC NHE-III₁ (nuclease hypersensitivity element III₁) “silencer” element. The G-Quadruplex structure is at the top-center, and the i-Motif is at the bottom-center of each picture. a “Rotation #1” of the G-Quadruplex, with the T15 loop at the top and rear and the G19/A20 loop at the top and front of the picture. b “Rotation #2” of the G-Quadruplex, with the T15 loop at the top and front of the image, and the G19/A20 loop at the front and adjacent to the G-Quadruplex/i-Motif interface     

The control of mitochondrial succinate-dependent H<Subscript>2</Subscript>O<Subscript>2</Subscript> production

In brain mitochondria succinate activates H₂O₂ release, concentration dependently (starting at 15 μM), and in the presence of NAD dependent substrates (glutamate, pyruvate, β-hydroxybutyrate). We report that TCA cycle metabolites (citrate, isocitrate, α-ketoglutarate, fumarate, malate) individually and quickly inhibit H₂O₂ release. When they are present together at physiological concentration (0.2, 0.01, 0.15, 0.12, 0.2 mM respectively) they decrease H₂O₂ production by over 60% at 0.1–0.2 mM succinate. The degree of inhibition depends on the concentration of each metabolite. Acetoacetate is a strong inhibitor of H₂O₂ release, starting at 10 μM and acting quickly. It potentiates the inhibition induced by TCA cycle metabolites. The action of acetoacetate is partially removed by β-hydroxybutyrate. Removal is minimal at 0.1 mM acetoacetate, and is higher at 0.5 mM acetoacetate. We conclude that several inhibitors of H₂O₂ release act jointly and concentration dependently to rapidly set the required level of H₂O₂ generation at each succinate concentration.     

Recent improvements in the development of A<Subscript>2B</Subscript> adenosine receptor agonists

Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A₁, A_2A, A_2B and A₃ (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A_2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A_2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A_2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N ⁶-, C²-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA₁ K _i = 1050 nM, hA_2A K _i = 1550 nM, hA_2B EC₅₀ = 82 nM, hA₃ K _i > 5 μM) and its 2-chloro analogue 23 (hA₁ K _i = 3500 nM, hA_2A K _i = 4950 nM, hA_2B EC₅₀ = 210 nM, hA₃ K _i > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA_2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA₁, hA_2A, hA₃ EC₅₀ > 10 μM; hA_2B EC₅₀ = 3 nM) is currently under preclinical-phase investigation for treating coronary artery disorders and atherosclerosis.     

Muscarinic Inhibition of Hippocampal and Striatal Adenylyl Cyclase is Mainly Due to the M<Subscript>4</Subscript> Receptor

The five muscarinic acetylcholine receptors (M₁–M₅) are differentially expressed in the brain. M₂ and M₄ are coupled to inhibition of stimulated adenylyl cyclase, while M₁, M₃ and M₅ are mainly coupled to the phosphoinositide pathway. We studied the muscarinic receptor regulation of adenylyl cyclase activity in the rat hippocampus, compared to the striatum and amygdala. Basal and forskolin-stimulated adenylyl cyclase activity was higher in the striatum but the muscarinic inhibition was much lower. Highly selective muscarinic toxins MT1 and MT2—affinity order M₁ ≥ M₄ >> others—and MT3—highly selective M₄ antagonist—did not show significant effects on basal or forskolin-stimulated cyclic AMP production but, like scopolamine, counteracted oxotremorine inhibition. Since MTs have negligible affinity for M₂, M₄ would be the main subtype responsible for muscarinic inhibition of forskolin-stimulated enzyme. Dopamine stimulated a small fraction of the enzyme (3.1% in striatum, 1.3% in the hippocampus). Since MT3 fully blocked muscarinic inhibition of dopamine-stimulated enzyme, M₄ receptor would be responsible for this regulation. Diana Jerusalinsky and Edgar Kornisiuk contributed equally to this paper.     

Ontogeny of chicken ductus arteriosus response to oxygen and vasoconstrictors

The present study aimed to characterize the contractile reactivity of the chicken ductus arteriosus (DA) from the last stage of prenatal development and throughout the perinatal period. Isolated DA rings from 15-day, noninternally-pipped 19-day, and externally-pipped 21-day embryos were studied using myograph techniques. On embryonic day 15, the chicken DA did not respond to O(2) (0 to 21%), norepinephrine (NE), or phenylephrine (Phe) but contracted in response to high-K(+) solution, the inhibitor of voltage-gated channels 4-aminopyridine, U-46619, and endothelin (ET)-1. These responses increased with advancing incubation age. Contractile responses to O(2), NE, and Phe were present in the 19- and 21-day embryo. Oxygen-induced contraction was restricted to the pulmonary side of the DA and was augmented by the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and reduced by the peptidic ET(A) and ET(B)-receptor antagonist PD-142,893. Transmural electrical stimulation of nerves, the nonselective cyclooxygenase (COX) inhibitor indomethacin, the COX-1 inhibitor valeryl salicylate, the COX-2 inhibitor nimesulide, the inhibitor of ATP-sensitive K(+) channels glibenclamide, and the inhibitor of Ca(2+)-activated K(+) channels tetraethylammonium did not cause contraction of the DA rings at any age. We conclude that transition to ex ovo life is accompanied by dramatic changes in chicken DA reactivity. At 0.7 incubation, excitation-contraction and pharmacomechanical coupling for several contractile agonists are already present, whereas the constrictor effects of O(2) and cathecolamines appear later in development and are located in the pulmonary side of the DA.     

Reduction of urate crystal-induced inflammation by root extracts from traditional oriental medicinal plants: elevation of prostaglandin D<Subscript>2</Subscript>levels

Dried roots of the plants Acanthopanax senticosus, Angelica sinensis and Scutellaria baicalensis are used in traditional oriental medicine and reportedly possess anti-inflammatory properties. Using the murine air pouch model of inflammation, we investigated the efficacy and mode of action of an extract from these three plants in crystal-induced inflammation. Air pouches were raised on the backs of 8-week-old BALB/c mice. Mice were fed 100 mg/kg body weight of root extracts (A. senticosus:A. sinensis:S. baicalensis mixed in a ratio of 5:4:1 by weight) or vehicle only on days 3–6. Inflammation was elicited on day 6 by injecting 2 mg of monosodium urate (MSU) crystals into the pouch. Neutrophil density and IL-6 and TNF-α mRNA levels were determined in the pouch membrane, and the leukocyte count and IL-6, prostaglandin E₂ (PGE₂) and prostaglandin D₂ (PGD₂) levels were determined in the pouch exudate. Treatment with the root extracts led to a reduction in all inflammatory parameters: the leukocyte count in the pouch exudate decreased by 82%; the neutrophil density in the pouch membrane decreased by 68%; IL-6 and TNF-α mRNA levels in the pouch membrane decreased by 100%; the IL-6 concentration in the pouch fluid decreased by 50%; and the PGE₂ concentration in the pouch fluid decreased by 69%. Remarkably, the concentration of the potentially anti-inflammatory PGD₂ rose 5.2-fold in the pouch exudate (p < 0.005), which led to a normalization of the PGD₂:PGE₂ ratio. A 3.7-fold rise in hematopoietic PGD synthase (h-PGDS) mRNA paralleled this rise in PGD₂ (p = 0.01).     

Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)γ agonists on membrane-associated prostaglandin E2 synthase-1 in IL-1β-stimulated rat chondrocytes: evidence for PPARγ-independent inhibition by 15-deoxy-Δ12,14prostaglandin J2

Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E₂ synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF_1α and PGE₂ in rat chondrocytes stimulated with 10 ng/ml IL-1β, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)γ agonists. Real-time PCR analysis showed that IL-1β induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF_1α and PGE₂ peaked 24 hours after stimulation with IL-1β; the induction of PGE₂ was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Δ^12,14prostaglandin J₂ (15d-PGJ₂) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 μM), with more potency on PGE₂ level than on 6-keto-PGF_1α level (-90% versus -66% at 10 μM). A high dose of 15d-PGJ₂ partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 μM. Inhibitory effects of 10 μM 15d-PGJ₂ were neither reduced by PPARγ blockade with GW-9662 nor enhanced by PPARγ overexpression, supporting a PPARγ-independent mechanism. EMSA and TransAM^? analyses demonstrated that mutated IκBα almost completely suppressed the stimulating effect of IL-1β on mPGES-1 expression and PGE₂ production, whereas 15d-PGJ₂ inhibited NF-κB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE₂ synthesis; second, activation of the prostaglandin cascade requires NF-κB activation; third, 15d-PGJ₂ strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ₂ occurs independently of PPARγ through inhibition of the NF-κB pathway; fifth, mPGES-1 is the main target of 15d-PGJ₂.