Inhibition of microsomal prostaglandin E2 synthase-1 as a molecular basis for the anti-inflammatory actions of boswellic acids from frankincense

BACKGROUND AND PURPOSE

Frankincense, the gum resin derived from Boswellia species, showed anti-inflammatory efficacy in animal models and in pilot clinical studies. Boswellic acids (BAs) are assumed to be responsible for these effects but their anti-inflammatory efficacy in vivo and their molecular modes of action are incompletely understood.

EXPERIMENTAL APPROACH

A protein fishing approach using immobilized BA and surface plasmon resonance (SPR) spectroscopy were used to reveal microsomal prostaglandin E₂ synthase-1 (mPGES1) as a BA-interacting protein. Cell-free and cell-based assays were applied to confirm the functional interference of BAs with mPGES1. Carrageenan-induced mouse paw oedema and rat pleurisy models were utilized to demonstrate the efficacy of defined BAs in vivo.

KEY RESULTS

Human mPGES1 from A549 cells or in vitro-translated human enzyme selectively bound to BA affinity matrices and SPR spectroscopy confirmed these interactions. BAs reversibly suppressed the transformation of prostaglandin (PG)H₂ to PGE₂ mediated by mPGES1 (IC₅₀ = 3–10 µM). Also, in intact A549 cells, BAs selectively inhibited PGE₂ generation and, in human whole blood, β-BA reduced lipopolysaccharide-induced PGE₂ biosynthesis without affecting formation of the COX-derived metabolites 6-keto PGF_1α and thromboxane B₂. Intraperitoneal or oral administration of β-BA (1 mg·kg⁻¹) suppressed rat pleurisy, accompanied by impaired levels of PGE₂ and β-BA (1 mg·kg⁻¹, given i.p.) also reduced mouse paw oedema, both induced by carrageenan.

CONCLUSIONS AND IMPLICATIONS

Suppression of PGE₂ formation by BAs via interference with mPGES1 contribute to the anti-inflammatory effectiveness of BAs and of frankincense, and may constitute a biochemical basis for their anti-inflammatory properties.     

Regulation of the inducible cyclo-oxygenase pathway in human cultured airway epithelial (A549) cells by nitric oxide

1. In airway epithelium, nitric oxide (NO) is synthesized in the setting of inflammation by inducible nitric oxide synthase (iNOS). Although the role of epithelial derived NO in the regulation of human airways is unknown, prostaglandin E₂ (PGE₂) is recognised as an important inhibitory mediator in human airways. Cyclo-oxygenase (COX) is the rate limiting enzyme in the production of prostanoids and since inflammatory pathways enhance the expression of an inducible COX (COX-2), both COX-2 and iNOS may be co-expressed in response to an inflammatory stimulus. Although regulation of the COX-2 pathway by NO has been demonstrated in animal models, its potential importance in human airway epithelium has not been investigated.
2. The effect of endogenous and exogenous NO on the COX-2 pathway was investigated in the A549 human airway epithelial cell culture model. Activity of the COX-2 pathway was assessed by PGE₂ EIA, and iNOS pathway activity by nitrite assay. A combination cytokine stimulus of interferon gamma (IFNγ) 100 u ml⁻¹, interleukin-1β (IL-1β) 1 u ml⁻¹ and lipopolysaccharide (LPS) 10 μg ml⁻¹ induced nitrite formation which could be inhibited by the competitive NOS inhibitor N^G-nitro-L-arginine-methyl-ester (L-NAME). IL-1β alone (1–50 u ml⁻¹) induced PGE₂ formation without significant nitrite formation, a response which was inhibited by the COX-2 specific inhibitor nimesulide. Submaximal stimuli used for further experiments were IFNγ 100 u ml⁻¹, IL-1β 1 u ml⁻¹ and LPS 10 μg ml⁻¹ to induce both the iNOS and COX-2 pathways, and IL-1β 3 u ml⁻¹ to induce COX-2 without iNOS activity.
3. Cells treated with IFNγ 100 u ml⁻¹, IL-1β 1 u ml⁻¹ and LPS 10 μg ml⁻¹ for 48 h either alone, or with the addition of L-NAME (0 to 10⁻² M), demonstrated inhibition by L-NAME of PGE₂ (3.61±0.55 to 0.51±0.04 pg/10⁴ cells; P<0.001) and nitrite (34.33±8.07 to 0 pmol/10⁴ cells; P<0.001) production. Restoration of the PGE₂ response (0.187±0.053 to 15.46±2.59 pg/10⁴ cells; P<0.001) was observed after treating cells with the same cytokine stimulus and L-NAME 10⁻⁶ M, but with the addition of the NOS substrate L-arginine (0 to 10⁻⁵ M).
4. Cells incubated with IL-1β 3 u ml⁻¹ for 6 h, either alone or with addition of the NO donor S-nitroso-acetyl-penicillamine (SNAP) (0 to 10⁻⁴ M), demonstrated increased PGE₂ formation (1.23±0.03 to 2.92±0.19 pg/10⁴ cells; P< 0.05). No increase in PGE₂ formation was seen when the experiment was repeated in the presence of the guanylate cyclase inhibitor methylene blue (50 μM). Cells treated with SNAP alone did not demonstrate an increased PGE₂ formation. Cells incubated with IL-1β 3 u ml⁻¹ for 6 h in the presence of dibutyryl cyclic guanylate monophosphate (0 to 10⁻³ M) also demonstrated an increased PGE₂ response (2.56±0.21 to 4.53±0.64 pg/10⁴ cells; P<0.05).
5. These data demonstrate that in a human airway epithelial cell culture system, both exogenous and endogenous NO increase the activity of the COX-2 pathway in the setting of inflammatory cytokine stimulation, and that this effect is likely to be mediated by guanylate cyclase. This suggests a role for NO in the regulation of human airway inflammation.

     

Microsomal prostaglandin E synthase-1 and cyclooxygenase-2 are both required for ischaemic excitotoxicity

Background and purpose:

Although both microsomal prostaglandin E synthase (mPGES)-1 and cyclooxygenase (COX)-2 are critical factors in stroke injury, but the interactions between these enzymes in the ischaemic brain is still obscure. This study examines the hypothesis that mPGES-1 activity is required for COX-2 to cause neuronal damage in ischaemic injury.

Experimental approach:

We used a glutamate-induced excitotoxicity model in cultures of rat or mouse hippocampal slices and a mouse middle cerebral artery occlusion-reperfusion model in vivo. The effect of a COX-2 inhibitor on neuronal damage in mPGES-1 knockout (KO) mice was compared with that in wild-type (WT) mice.

Key results:

In rat hippocampal slices, glutamate-induced excitotoxicity, as well as prostaglandin (PG) E₂ production and PGES activation, was significantly attenuated by either MK-886 or NS-398, inhibitors of mPGES-1 and COX-2 respectively; however, co-application of these inhibitors had neither an additive nor a synergistic effect. The protective effect of NS-398 on the excitotoxicity observed in WT slices was completely abolished in mPGES-1 KO slices, which showed less excitotoxicity than WT slices. In the transient focal ischaemia model, mPGES-1 and COX-2 were co-localized in the infarct region of the cortex. Injection of NS-398 reduced not only ischaemic PGE₂ production, but also ischaemic injuries in WT mice, but not in mPGES-1 KO mice, which showed less dysfunction than WT mice.

Conclusion and implications:

Microsomal prostaglandin E synthase-1 and COX-2 are co-induced by excess glutamate in ischaemic brain. These enzymes are co-localized and act together to exacerbate stroke injury, by excessive PGE₂ production.     

Inotropy and L-type Ca2+ current,activated by β1- and β2-adrenoceptors,are differently controlled by phosphodiesterases 3 and 4 in rat heart

Background and purpose

β₁- and β₂-adrenoceptors coexist in rat heart but β₂-adrenoceptor-mediated inotropic effects are hardly detectable, possibly due to phosphodiesterase (PDE) activity. We investigated the influence of the PDE3 inhibitor cilostamide (300 nmol·L⁻¹) and the PDE4 inhibitor rolipram (1 µmol·L⁻¹) on the effects of (−)-catecholamines.

Experimental approach

Cardiostimulation evoked by (−)-noradrenaline (ICI118551 present) and (−)-adrenaline (CGP20712A present) through β₁- and β₂-adrenoceptors, respectively, was compared on sinoatrial beating rate, left atrial and ventricular contractile force in isolated tissues from Wistar rats. L-type Ca²⁺-current (I_Ca-L) was assessed with whole-cell patch clamp.

Key results

Rolipram caused sinoatrial tachycardia. Cilostamide and rolipram did not enhance chronotropic potencies of (−)-noradrenaline and (−)-adrenaline. Rolipram but not cilostamide potentiated atrial and ventricular inotropic effects of (−)-noradrenaline. Cilostamide potentiated the ventricular effects of (−)-adrenaline but not of (−)-noradrenaline. Concurrent cilostamide + rolipram uncovered left atrial effects of (−)-adrenaline. Both rolipram and cilostamide augmented the (−)-noradrenaline (1 µmol·L⁻¹) evoked increase in I_Ca-L. (−)-Adrenaline (10 µmol·L⁻¹) increased I_Ca-L only in the presence of cilostamide but not rolipram.

Conclusions and implications

PDE4 blunts the β₁-adrenoceptor-mediated inotropic effects. PDE4 reduces basal sinoatrial rate in a compartment distinct from compartments controlled by β₁- and β₂-adrenoceptors. PDE3 and PDE4 jointly prevent left atrial β₂-adrenoceptor-mediated inotropy. Both PDE3 and PDE4 reduce I_Ca-L responses through β₁-adrenoceptors but the PDE3 component is unrelated to inotropy. PDE3 blunts both ventricular inotropic and I_Ca-L responses through β₂-adrenoceptors.     

Investigation of the different adrenoceptor targets of nebivolol enantiomers in rat thoracic aorta

Background and purpose:

Nebivolol is a highly selective β₁-adrenoceptor antagonist with β₃-adrenoceptor agonist properties and is a racemate mixture of D- and L-enantiomers. However, the cellular mechanisms of the effects of each enantiomer are not yet clear and are a matter for debate. The aim of the present experiments was to determine the adrenoceptors involved in the vascular effects of D- and L-enantiomers of nebivolol in rat thoracic aorta.

Experimental approach:

Responses to nebivolol enantiomers were evaluated in rings of thoracic aorta from male Sprague-Dawley rats.

Key results:

D-nebivolol (0.1–10 µmol·L⁻¹), but not L-nebivolol, significantly shifted to the right the concentration-response curve to phenylephrine, an α₁-adrenoceptor agonist, in a concentration-dependent manner. For the following experiments, aortic rings were constricted with endothelin 1 and now both enantiomers produced an endothelium-dependent relaxation of the rings involving the nitric oxide pathway. This relaxation was not modified by 1 µmol·L⁻¹ CGP 20,712A (β₁-adrenoceptor antagonist), but significantly blunted by 7 µmol·L⁻¹ L-74,8337 (β₃-adrenoceptor antagonist). However, only the vasorelaxation induced by D-nebivolol was significantly reduced by 1 µmol·L⁻¹ ICI 118,551 (β₂-adrenoceptor antagonist).

Conclusions and implications:

Our results suggest that the nebivolol enantiomers act on different targets. D-nebivolol induced vasorelaxation by activating β₂- and β₃-adrenoceptors and antagonizing α₁-adrenoceptors. L-nebivolol induced vasorelaxation by activating only β₃-adrenoceptors in our model. Our results emphasize that nebivolol is a β₁-adrenoceptor antagonist with several important pharmacological differences from other β₁-adrenoceptor antagonists.     

Inhibition of colonic motility and defecation by RS-127445 suggests an involvement of the 5-HT2B receptor in rodent large bowel physiology

Background:

5-HT_2B receptors are localized within the myenteric nervous system, but their functions on motor/sensory neurons are unclear. To explore the role of these receptors, we further characterized the 5-HT_2B receptor antagonist RS-127445 and studied its effects on peristalsis and defecation.

Experimental approach:

Although reported as a selective 5-HT_2B receptor antagonist, any interactions of RS-127445 with 5-HT₄ receptors are unknown; this was examined using the recombinant receptor and Biomolecular Interaction Detection technology. Mouse isolated colon was mounted in tissue baths for isometric recording of neuronal contractions evoked by electrical field stimulation (EFS), or under an intraluminal pressure gradient to induce peristalsis; the effects of RS-127445 on EFS-induced and on peristaltic contractions were measured. Faecal output of rats in grid-bottom cages was measured over 3 h following i.p. RS-127445 and separately, validation of the effective doses was achieved by determining the free, unbound fraction of RS-127445 in blood and brain.

Key results:

RS-127445 (up to 1 µmol·L⁻¹) did not interact with the 5-HT₄ receptor. RS-127445 (0.001–1 µmol·L⁻¹) did not affect EFS-induced contractions of the colon, although at 10 µmol·L⁻¹ the contractions were reduced (to 36 ± 8% of control, n= 4). RS-127445 (0.1–10 µmol·L⁻¹) concentration-dependently reduced peristaltic frequency (n= 4). RS-127445 (1–30 mg·kg⁻¹), dose-dependently reduced faecal output, reaching significance at 10 and 30 mg·kg⁻¹ (n= 6–11). In blood and brain, >98% of RS-127445 was protein-bound.

Conclusions and implications:

High-protein binding of RS-127445 indicates that relatively high doses are required for efficacy. The results suggest that 5-HT_2B receptors tonically regulate colonic motility.     

Prostanoid EP1 receptor as the target of (?)-epigallocatechin-3-gallate in suppressing hepatocellular carcinoma cells in vitro

Aim:

To investigate the effects of (−)-epigallocatechin-3-gallate (EGCG), an active compound in green tea, on prostaglandin E₂ (PGE₂)-induced proliferation and migration, and the expression of prostanoid EP₁ receptors in hepatocellular carcinoma (HCC) cells.

Methods:

HCC cell line HepG2, human hepatoma cell lines MHCC-97L, MHCC-97H and human hepatocyte cell line L02 were used. Cell viability was analyzed using MTT assay. PGE₂ production was determined with immunoassay. Wound healing assay and transwell filter assay were employed to assess the extent of HCC cell migration. The expression of EP₁ receptor and Gq protein were examined using Western blot assay.

Results:

PGE₂ (4-40000 nmol/L) or the EP₁ receptor agonist ONO-DI-004 (400-4000 nmol/L) increased the viability and migration of HepG2 cells in concentration-dependent manners. EGCG (100 μg/mL) significantly inhibited the viability and migration of HepG2 cells induced by PGE₂ or ONO-DI-004. HepG2 cells secreted an abundant amount of PGE₂ into the medium, and EGCG (100 μg/mL) significantly inhibited the PGE₂production and EP₁ receptor expression in HepG2 cells. EGCG (100 μg/mL) also inhibited the viability of MHCC-97L cells, but not that of MHCC-97H cells. Both EGCG (100 μg/mL) and EP₁ receptor antagonist ONO-8711 inhibited PGE₂ 4 μmol/L and ONO-DI-004 400 nmol/L-induced growth and migration of HepG2 cells. Both EGCG (100 μg/mL) and ONO-8711 210 nmol/L inhibited PGE₂- and ONO-DI-004-induced EP₁ expression. EGCG and ONO-8711 had synergistic effects in inhibiting EP₁ receptor expression. PGE₂, ONO-DI-004, ONO-8711, and EGCG had no effects on Gq expression in HepG2 cells, respectively.

Conclusion:

These findings suggest that the anti-HCC effects of EGCG might be mediated, at least partially, through the suppressing EP₁ receptor expression and PGE₂ production.     

Diadenosine pentaphosphate is a potent activator of cardiac ryanodine receptors revealing a novel high-affinity binding site for adenine nucleotides

Background and purpose:

Diadenosine polyphosphates are normally present in cells at low levels, but significant increases in concentrations can occur during cellular stress. The aim of this study was to investigate the effects of diadenosine pentaphosphate (Ap5A) and an oxidized analogue, oAp5A on the gating of sheep cardiac ryanodine receptors (RyR2).

Experimental approach:

RyR2 channel function was monitored after incorporation into planar bilayers under voltage-clamp conditions.

Key results:

With10 µmol·L⁻¹ cytosolic Ca²⁺, a significant ‘hump’ or plateau at the base of the dose–response relationship to Ap5A was revealed. Open probability (Po) was significantly increased to a plateau of approximately 0.2 in the concentration range 100 pmol·L⁻¹–10 µmol·L⁻¹. High Po values were observed at >10 µmol·L⁻¹ Ap5A, and Po values close to 1 could be achieved. Nanomolar levels of ATP and adenosine also revealed a hump at the base of the dose–response relationships, although GTP did not activate at any concentration, indicating a common, high-affinity binding site on RyR2 for adenine-based compounds. The oxidized analogue, oAp5A, did not significantly activate RyR2 via the high-affinity binding site; however, it could fully open the channel with an EC₅₀ of 16 µmol·L⁻¹ (Ap5A EC₅₀ = 140 µmol·L⁻¹). Perfusion experiments suggest that oAp5A and Ap5A dissociate slowly from their binding sites on RyR2.

Conclusions and implications:

The ability of Ap5A compounds to increase Po even in the presence of ATP and their slow dissociation from the channel may enable these compounds to act as physiological regulators of RyR2, particularly under conditions of cellular stress.     

Tachykinin receptor modulation of cyclooxygenase-2 expression in human polymorphonuclear leucocytes

Background and purpose:

We investigated the ability of natural and synthetic selective NK receptors agonists and antagonists to modulate cyclooxygenase-2 (COX-2) expression in human polymorphonuclear leucocytes (PMNs).

Experimental approach:

The presence of all three tachykinin in PMNs was assessed by Western blot and PCR techniques. Natural and synthetic ligands selective for the tachykinin receptors were used to modulate COX-2 protein (measured with Western blotting) and activity [as prostaglandin E₂ (PGE₂) output]. Effects of substance P (SP) on phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) activation were studied to analyse the signalling pathway involved in COX-2 up-regulation mediated by SP.

Key results:

Stimulation of NK receptors with the natural ligands SP, neurokinin A (NKA) and neurokinin B, in the pmol·L⁻¹-µmol·L⁻¹ concentration range, modulated COX-2 expression and PGE₂ release in a concentration- and time-dependent manner. Experiments with synthetic selective agonists [Sar⁹, Met(O₂)¹¹]SP, [β-Ala⁸] NKA(4-10), senktide or selective antagonists L703,606, SR48,968 or SR142801, confirmed that COX-2 up-regulation was mediated by NK receptors. We found that mainly p38, p42 and p46 MAPKs were phosphorylated by SP and SB202190, PD98059 and SP600125, which are selective inhibitors of these kinases, blocked SP-induced COX-2 expression. SP also induced nuclear translocation of NF-κB concentration-dependently, with a maximum effect at 1 nmol·L⁻¹.

Conclusions and implications:

Human PMNs possess functional NK₁, NK₂ and NK₃ receptors, which mediate the induction of COX-2 expression and NF-κB activation by SP.     

Stimulation of angiotensin AT2 receptors by the non-peptide agonist,Compound 21, evokes vasodepressor effects in conscious spontaneously hypertensive rats

Background and purpose:

Angiotensin type 2 receptor (AT₂ receptor) stimulation evokes vasodilator effects in vitro and in vivo that oppose the vasoconstrictor effects of angiotensin type 1 receptors (AT₁ receptors). Recently, a novel non-peptide AT₂ receptor agonist, Compound 21, was described, which exhibited high AT₂ receptor selectivity.

Experimental approach:

Functional cardiovascular effects of the drug candidate Compound 21 were assessed, using mouse isolated aorta and rat mesenteric arteries in vitro and in conscious spontaneously hypertensive rats (SHR).

Key results:

Compound 21 evoked dose-dependent vasorelaxations in aortic and mesenteric vessels, abolished by the AT₂ receptor antagonist, PD123319. In vivo, Compound 21 administered alone, at doses ranging from 50 to 1000 ng·kg⁻¹·min⁻¹ over 4 h did not decrease blood pressure in conscious normotensive Wistar-Kyoto rats or SHR. However, when given in combination with the AT₁ receptor antagonist, candesartan, Compound 21 (300 ng·kg⁻¹·min⁻¹) lowered blood pressure in SHR only. Further analysis in separate groups of conscious SHR revealed that, at a sixfold lower dose, Compound 21 (50 ng·kg⁻¹·min⁻¹) still evoked a significant depressor response in adult SHR (∼30 mmHg) when combined with different doses of candesartan (0.01 or 0.1 mg·kg⁻¹). Moreover, the Compound 21-evoked depressor effect was abolished when co-infused (50 µg·kg⁻¹·min⁻¹ for 2 h) with the AT₂ receptor antagonist PD123319.

Conclusion and implications:

Collectively, our results indicate that acute administration of Compound 21 evoked blood pressure reductions via AT₂ receptor stimulation. Thus Compound 21 can be considered an excellent drug candidate for further study of AT₂ receptor function in cardiovascular disease.     

Montelukast inhibits neutrophil pro-inflammatory activity by a cyclic AMP-dependent mechanism

Background and purpose

The objective of this study was to characterize the effects of the cysteinyl leukotriene receptor antagonist, montelukast (0.1–2 µmol·L⁻¹), on Ca²⁺-dependent pro-inflammatory activities, cytosolic Ca²⁺ fluxes and intracellular cAMP in isolated human neutrophils activated with the chemoattractants, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (1 µmol·L⁻¹) and platelet-activating factor (200 nmol·L⁻¹).

Experimental approach

Generation of reactive oxygen species was measured by lucigenin- and luminol-enhanced chemiluminescence, elastase release by a colourimetric assay, leukotriene B₄ and cAMP by competitive binding ELISA procedures, and Ca²⁺ fluxes by fura-2/AM-based spectrofluorimetric and radiometric (⁴⁵Ca²⁺) procedures.

Key results

Pre-incubation of neutrophils with montelukast resulted in dose-related inhibition of the generation of reactive oxygen species and leukotriene B₄ by chemoattractant-activated neutrophils, as well as release of elastase, all of which were maximal at 2 µmol·L⁻¹ (mean percentages of the control values of 30 ± 1, 12 ± 3 and 21 ± 3 respectively; P < 0.05). From a mechanistic perspective, treatment of chemoattractant-activated neutrophils with montelukast resulted in significant reductions in both post-peak cytosolic Ca²⁺ concentrations and store-operated Ca²⁺ influx. These montelukast-mediated alterations in Ca²⁺ handling by the cells were associated with a significant elevation in basal cAMP levels, which resulted from inhibition of cyclic nucleotide phosphodiesterases.

Conclusions and implications

Montelukast, primarily a cysteinyl leukotriene (CysLT₁) receptor antagonist, exhibited previously undocumented, secondary, neutrophil-directed anti-inflammatory properties, which appeared to be cAMP-dependent.     

Modelling of α1-adrenoceptor-mediated temporal dynamics of inotropic response in rat heart to assess ligand binding and signal transduction parameters

Background and purpose:

In order to use the transient response to an antagonist (prazosin) to evaluate properties of agonist interactions with the α₁-adrenoceptor system, an integrative mechanistic model of cardiac uptake of prazosin and its competitive interaction with phenylephrine at the receptor site was developed. Based on the operational model of agonism, the aim was to evaluate both the receptor binding and signal transduction process as determinants of the inotropic effect of phenylephrine.

Experimental approach:

In Langendorff-perfused rat hearts, prazosin outflow concentration and left ventricular developed pressure were measured, first in the presence of 12.3 µmol·L⁻¹ phenylephrine following a 1 min infusion of 1.27 nmol [³H]-prazosin, and second, when after 30 min the phenylephrine concentration in perfusate was reduced to 6.1 µmol·L⁻¹, the 1 min infusion of 1.27 nmol [³H]-prazosin was repeated.

Key results:

The kinetic model accounted for cardiac uptake and receptor binding kinetics of prazosin (dissociation constant, mean ± SD: 0.057 ± 0.012 nmol·L⁻¹), assuming that the competitive displacement of phenylephrine (dissociation constant: 101 ± 13 nmol·L⁻¹) reduced the receptor occupation by the agonist and, consequently, contractility. This competitive binding process appeared to be the rate-determining step in response generation. The relationship between receptor occupancy and inotropic response was described by an efficacy parameter (τ, ratio of receptor density and coupling efficiency) of 4.9.

Conclusions and implications:

Mechanistic pharmacodynamic modelling of the kinetics of antagonism by prazosin allows quantitative assessment of the α₁-adrenoceptor system both at the receptor and post-receptor levels.     

The antipyretic effect of dipyrone is unrelated to inhibition of PGE(2) synthesis in the hypothalamus

BACKGROUND AND PURPOSE

Bacterial lipopolysaccharide (LPS) induces fever through two parallel pathways; one, prostaglandin (PG)-dependent and the other, PG-independent and involving endothelin-1 (ET-1). For a better understanding of the mechanisms by which dipyrone exerts antipyresis, we have investigated its effects on fever and changes in PGE₂ content in plasma, CSF and hypothalamus induced by either LPS or ET-1.

EXPERIMENTAL APPROACH

Rats were given (i.p.) dipyrone (120 mg·kg⁻¹) or indomethacin (2 mg·kg⁻¹) 30 min before injection of LPS (5 µg·kg⁻¹, i.v.) or ET-1 (1 pmol, i.c.v.). Rectal temperature was measured by tele-thermometry. PGE₂ levels were determined in the plasma, CSF and hypothalamus by elisa.

KEY RESULTS

LPS or ET-1 induced fever and increased CSF and hypothalamic PGE₂ levels. Two hours after LPS, indomethacin reduced CSF and hypothalamic PGE₂ but did not inhibit fever, while at 3 h it reduced all three parameters. Three hours after ET-1, indomethacin inhibited the increase in CSF and hypothalamic PGE₂ levels but did not affect fever. Dipyrone abolished both the fever and the increased CSF PGE₂ levels induced by LPS or ET-1 but did not affect the increased hypothalamic PGE₂ levels. Dipyrone also reduced the increase in the venous plasma PGE₂ concentration induced by LPS.

CONCLUSIONS AND IMPLICATIONS

These findings confirm that PGE₂ does not play a relevant role in ET-1-induced fever. They also demonstrate for the first time that the antipyretic effect of dipyrone was not mechanistically linked to the inhibition of hypothalamic PGE₂ synthesis.     

Characterization of biosynthesis and modes of action of prostaglandin E2 and prostacyclin in guinea pig mesenteric lymphatic vessels

Background and purpose:

Rhythmical transient constrictions of the lymphatic vessels provide the means for efficient lymph drainage and interstitial tissue fluid balance. This activity is critical during inflammation, to avoid or limit oedema resulting from increased vascular permeability, mediated by the release of various inflammatory mediators. In this study, we investigated the mechanisms by which prostaglandin E₂ (PGE₂) and prostacyclin modulate lymphatic contractility in isolated guinea pig mesenteric lymphatic vessels.

Experimental approach:

Quantitative RT-PCR was used to assess the expression of mRNA for enzymes and receptors involved in the production and action of PGE₂ and prostacyclin in mesenteric collecting lymphatic vessels. Frequency and amplitude of lymphatic vessel constriction were measured in the presence of these prostaglandins and the role of their respective EP and IP receptors assessed.

Key results:

Prostaglandin E₂ and prostacyclin decreased concentration-dependently the frequency, without affecting the amplitude, of lymphatic constriction. Data obtained in the presence of the EP₄ receptor antagonists, GW627368x (1 µM) and AH23848B (30 µM) and the IP receptor antagonist CAY10441 (0.1 µM) suggest that PGE₂ predominantly activates EP₄, whereas prostacyclin mainly stimulates IP receptors. Inhibition of responses to either prostaglandin with H89 (10 µM) or glibenclamide (1 µM) suggested a role for the activation of protein kinase A and ATP-sensitive K⁺ channels.

Conclusions and implications:

Our findings characterized the inhibition of lymphatic pumping induced by PGE₂ or prostacyclin in guinea pig mesenteric lymphatics. This action is likely to impair oedema resolution and to contribute to the pro-inflammatory actions of these prostaglandins.     

NCX 2057, a novel NO-releasing derivative of ferulic acid,suppresses inflammatory and nociceptive responses in in vitro and in vivo models

Background and purpose:

We previously reported that NCX 2057, a compound comprising a nitric oxide (NO)-releasing moiety and the natural antioxidant, ferulic acid (FA), inhibits pro-inflammatory mediators through NO-mediated gene regulation. Here, we have assessed the activities of NCX 2057 in models of inflammatory and neuropathic pain, and characterized its effects on cyclooxygenase (COX)-1 and COX-2.

Experimental approach:

Anti-nociceptive and anti-inflammatory activities of NCX 2057 were measured in vitro and in vivo in models of inflammatory (carrageenan) and neuropathic (chronic constriction injury; CCI) pain. Effects of NCX 2057 were measured on COX-1 and COX-2 activities in RAW 264.7 macrophages.

Key results:

NCX 2057 dose-dependently inhibited single motor unit responses to noxious mechanical stimulation (ID₅₀= 100 µmol·kg⁻¹) and wind-up responses in rats with paw inflammation induced by carrageenan. Moreover, NCX 2057 inhibited allodynic responses following CCI of the sciatic nerve [ipsilateral Paw Withdrawal Threshold (g): vehicle: 41.4 ± 3.3; NCX 2057: 76.3 ± 4.8 FA: 37.9 ± 15.5 at 175 µmol·kg⁻¹]. NCX 2057 reversed carrageenan-induced hyperalgesic responses in mice and inhibited prostaglandin E₂ formation in paw exudates. Finally, NCX 2057 competitively inhibited COX-1 and COX-2 activities in whole RAW macophages (IC₅₀= 14.7 ± 7.4 and 21.6 ± 7.5 µM, respectively). None of these properties were exhibited by equivalent treatments with FA or standard NO donor compounds.

Conclusions and implications:

These studies indicate that NCX 2057 is effective in chronic inflammatory and neuropathic pain models, probably because of its particular combination of anti-COX, antioxidant and NO-releasing properties.     

Quercetin induces insulin secretion by direct activation of L-type calcium channels in pancreatic beta cells

Background and Purpose

Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca²⁺]_i) in beta cells, in the absence of any co-stimulating factor.

Experimental Approach

Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca²⁺]_i were measured using the ratiometric fluorescent Ca²⁺ indicator Fura-2. Ca²⁺ channel currents were recorded with the whole-cell patch-clamp technique.

Key Results

Quercetin concentration-dependently increased insulin secretion and elevated [Ca²⁺]_i. These effects were not modified by the SERCA inhibitor thapsigargin (1 μmol·L⁻¹), but were nearly abolished by the L-type Ca²⁺ channel antagonist nifedipine (1 μmol·L⁻¹). Similar to the L-type Ca²⁺ channel agonist Bay K 8644, quercetin enhanced the L-type Ca²⁺ current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca²⁺]_i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 μmol·L⁻¹), with the two drugs having cumulative effects on [Ca²⁺]_i.

Conclusions and Implications

Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca²⁺ influx through an interaction with L-type Ca²⁺ channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin''s mechanism of action on insulin secretion.     

Role of α1- and β3-adrenoceptors in the modulation by SR59230A of the effects of MDMA on body temperature in the mouse

Background and purpose:

We have investigated the ability of the β₃-adrenoceptor antagonist 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4,-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride (SR59230A) to affect the hyperthermia produced by methylenedioxymethamphetamine (MDMA) in conscious mice and whether α₁-adrenoceptor antagonist actions are involved.

Experimental approach:

Mice were implanted with temperature probes under anaesthesia, and allowed 2 week recovery. MDMA (20 mg·kg⁻¹) was administered subcutaneously 30 min after vehicle or test antagonist and effects on body temperature monitored by telemetry.

Key results:

Following vehicle, MDMA produced a slowly developing hyperthermia, reaching a maximum increase of 1.8°C at 130 min post injection. A low concentration of SR59230A (0.5 mg·kg⁻¹) produced a small but significant attenuation of the slowly developing hyperthermia to MDMA. A high concentration of SR59230A (5 mg·kg⁻¹) revealed a significant and marked early hypothermic reaction to MDMA, an effect that was mimicked by the α₁-adrenoceptor antagonist prazosin. Functional and ligand binding studies revealed actions of SR59230A at α₁-adrenoceptors.

Conclusions and implications:

1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4,-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride in high concentrations modulates the hyperthermic actions of MDMA in mice in two ways: by blocking an early α₁-adrenoceptor-mediated component to reveal a hypothermia, and by a small attenuation of the later hyperthermic component which may possibly be β₃-adrenoceptor-mediated (this seen with the low concentration of SR59230A). Hence, the major actions of SR59230A in modulating the actions of MDMA on temperature involve α₁-adrenoceptor antagonism.