Lipoxin A4 suppresses the development of endometriosis in an ALXreceptor-dependent manner via the p38 MAPK pathway

Background and Purpose

Lipoxins can function as endogenous ‘breaking signals’ in inflammation and play important roles in the progression of endometriosis. In this study, we further investigated the molecular mechanism by which lipoxin A₄ (LXA₄) suppresses the development of endometriosis.

Experimental Approach

Primary endometriotic stromal cells (ESCs) were treated with IL-1β, or pre-incubated with LXA₄ before incubation with IL-1β. The LXA₄ receptor (ALX receptor) antagonist Boc-2 and gene-silencing approaches were used to study the involvement of the ALX receptor in anti-inflammatory signalling responses in ESCs. An animal model of endometriosis was induced in BALB/c mice by i.p. injection of an endometrium-rich fragment.

Key Results

Decreased levels of LXA₄ and 15-LOX-2 expression but increased expression of AXL receptors were observed in endometriotic tissues. LXA₄ inhibited the release of inflammatory factors and phosphorylation of p38 MAPK in IL-1β-induced ESCs, an effect mediated by ALX receptors. LXA₄ inhibited the proliferation of ESCs, as indicated by reduced DNA replication, caused G₀/G₁ phase cell cycle arrest and down-regulated the expression of proliferating cell nuclear antigen in ESCs. LXA₄ also attenuated the invasive activity of ESCs mainly by suppressing the expression and activity of MMP-9. In vivo, we further confirmed that LXA₄ could inhibit the progression of endometriosis by acting as an anti-inflammatory.

Conclusions and Implications

LXA₄ exerted anti-inflammatory, anti-proliferative and anti-invasive effects on endometriosis through a mechanism that involved down-regulating the activities of p38 MAPK, which was mediated by ALX receptors.     

Heterologously expressed formyl peptide receptor 2 (FPR2/ALX) does not respond to lipoxin A4

Lipoxin A₄ (LXA₄) has been described as an anti-inflammatory mediator, which exerts its effects through the formyl peptide receptor FPR2, also known as ALX. However, there has been a controversy whether or not cells expressing FPR2/ALX, such as neutrophils, respond to LXA₄. We, therefore, systematically examined the ability of the human and murine forms of the receptor to respond to LXA₄. We show that both receptor orthologues responded to the FPR2/ALX peptide agonist WKYMVM when expressed heterologously. In contrast, LXA₄ from different sources neither increased [Ca²⁺]_i and extracellular-signal-regulated kinase (ERK) phosphorylation, nor did it induce a decrease in cAMP levels or a translocation of β-arrestin. Also, several LXA₄ analogs were found to be unable to signal through FPR2/ALX. We conclude that FPR2/ALX is not activated by LXA₄ and that the molecular mechanism by which LXA₄ functions still needs to be identified.     

Effect of plant neutrophil elastase inhibitor on leucocyte migration, adhesion and cytokine release in inflammatory conditions

BACKGROUND AND PURPOSE

The serine and cysteine peptidase inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, is similar to the classical plant Kunitz inhibitor, STI, but lacks disulphide bridges and methionine residues. BbCI blocks activity of the serine peptidases, elastase (K_iapp 5.3 nM) and cathepsin G (K_iapp 160.0 nM), and the cysteine peptidase cathepsin L (K_iapp 0.2 nM). These three peptidases play important roles in the inflammatory process.

EXPERIMENTAL APPROACH

We measured the effects of BbCI on paw oedema and on leucocyte accumulation in pleurisy, both induced by carrageenan. Leucocyte–endothelial cell interactions in scrotal microvasculature in Wistar rats were investigated using intravital microscopy. Cytokine levels in pleural exudate and serum were measured by elisa.

KEY RESULTS

Pretreatment of the animals with BbCI (2.5 mg·kg⁻¹), 30 min before carrageenan-induced inflammation, effectively reduced paw oedema and bradykinin release, neutrophil migration into the pleural cavity. The number of rolling, adhered and migrated leucocytes at the spermatic fascia microcirculation following carrageenan injection into the scrotum were reduced by BbCI pretreatment. Furthermore, levels of the rat chemokine cytokine-induced neutrophil chemo-attractant-1 were significantly reduced in both pleural exudates and serum from animals pretreated with BbCI. Levels of interleukin-1β or tumour necrosis factor-α, however, did not change.

CONCLUSIONS AND IMPLICATIONS

Taken together, our data suggest that the anti-inflammatory properties of BbCI may be useful in investigations of other pathological processes in which human neutrophil elastase, cathepsin G and cathepsin L play important roles.     

In vitro and in vivo characterization of A-940894: a potent histamine H4 receptor antagonist with anti-inflammatory properties

Background and purpose:

The histamine H₄ receptor is widely expressed in cells of immune origin and has been shown to play a role in a variety of inflammatory processes mediated by histamine. In this report, we describe the in vitro and in vivo anti-inflammatory properties of a potent histamine H₄ receptor antagonist, A-940894 (4-piperazin-1-yl-6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-d]pyrimidin-2-ylamine).

Experimental approach:

We have analysed the pharmacological profile of A-940894 at mouse native, rat recombinant and human recombinant and native, histamine H₄ receptors by radioligand binding, calcium mobilization, mast cell shape change, eosinophil chemotaxis assays and in the mouse model of zymosan-induced peritonitis.

Key results:

A-940894 potently binds to both human and rat histamine H₄ receptors and exhibits considerably lower affinity for the human histamine H₁, H₂ or H₃ receptors. It potently blocked histamine-evoked calcium mobilization in the fluorometric imaging plate reader assays and inhibited histamine-induced shape change of mouse bone marrow-derived mast cells and chemotaxis of human eosinophils in vitro. In a mouse mast cell-dependent model of zymosan-induced peritonitis, A-940894 significantly blocked neutrophil influx and reduced intraperitoneal prostaglandin D₂ levels. Finally, A-940894 has good pharmacokinetic properties, including half-life and oral bioavailability in rats and mice.

Conclusions and Implications:

These data suggest that A-940894 is a potent and selective histamine H₄ receptor antagonist with pharmacokinetic properties suitable for long-term in vivo testing and could serve as a useful tool for the further characterization of histamine H₄ receptor pharmacology.     

Anti-inflammatory synergy of MEN16132, a kinin B2 receptor antagonist, and dexamethasone in carrageenan-induced knee joint arthritis in rats

BACKGROUND AND PURPOSE

Bradykinin, through its B₂ receptor, is involved in inflammatory processes related to arthropathies. In carrageenan and lipopolysaccharide (LPS)-induced arthritis in rat, the anti-inflammatory activity of MEN16132, a potent and selective kinin B₂ receptor antagonist, was compared with that of steroidal and nonsteroidal anti-inflammatory drugs. The interaction between MEN16132 and dexamethasone was also investigated.

EXPERIMENTAL APPROACH

Drugs, alone or in combination, were injected into the knee joint 30 min before intra-articular administration of carrageenan or LPS, in pentobarbital anaesthetized rats. Effects on incapacitation, oedema, neutrophil recruitment and kallikrein system activation, in the knee joint, were assessed.

KEY RESULTS

MEN16132 and dexamethasone (10–300 µg per knee) dose-dependently reduced carrageenan-induced joint pain, oedema and neutrophil infiltration, reaching a maximal inhibition of about 50%. Dexketoprofen exerted a similar analgesic activity, whereas it did not affect the other inflammatory responses. MEN16132 showed a partial inhibition of LPS-induced joint pain, whereas dexamethasone produced a full analgesic effect. Combination of MEN16132 and dexamethasone showed a strong synergistic interaction in inhibiting both carrageenan and LPS-induced knee joint inflammation. Dexamethasone did not prevent the contact activation of prekallikrein by carrageenan and the subsequent release of kallikreins and bradykinin in the synovium.

CONCLUSIONS AND IMPLICATIONS

Steroids and kinin B₂ receptor antagonists appear to relieve arthritic symptoms induced by carrageenan or LPS and act synergistically to inhibit joint inflammation. This could have interesting therapeutic implications, possibly opening the way for combination therapies in the control of inflammatory arthropathies.     

Glucocorticoid-stimulated,transcription-independent release of annexin A1 by cochlear Hensen cells

Background and purpose:

The current clinical strategy to protect the auditory organ against inflammatory damage by migrating leukocytes is the local delivery of glucocorticoids. However, the mechanism by which glucocorticoids confer this protection remains unknown. Therefore, we investigated the cellular and molecular targets of glucocorticoids in the cochlea that could be involved in preventing leukocyte migration.

Experimental approach:

We used microscopy as well as immunocytochemical and microfluidic techniques to elucidate the effect of dexamethasone, hydrocortisone and prednisolone on the cellular and intracellular distribution of annexin A1 (ANXA1) – a glucocorticoid target known to inhibit leukocyte migration by receptor-mediated signalling – in the cochlea and isolated cochlear cells of guinea pigs.

Key results:

All the cells lining the scala media – the cochlear compartment containing the auditory organ – express ANXA1 and the ANXA1 receptor FPR2/ALX is present in the scala media, as well as in other cochlear ducts. The majority of ANXA1 in the scala media is stored inside lipid droplets within cochlear Hensen cells. Glucocorticoids activate a myosin IIC-mediated mechanism that drives ANXA1 from the lipid droplets to the apical region of the Hensen cells, where ANXA1 is released to the external milieu by a process involving ABC transporters.

Conclusions and implications:

These findings suggest that ANXA1 could be a major mediator of the anti-inflammatory effects of glucocorticoids in the cochlea and identify new molecular targets for prevention of sudden sensorineural hearing loss.     

Rimonabant reduces keratinocyte viability by induction of apoptosis and exerts topical anti-inflammatory activity in mice

BACKGROUND AND PURPOSE

There is growing evidence that the cannabinoid CB₁ receptor antagonist, rimonabant (SR141716) exerts potential anti-proliferative and anti-inflammatory actions. Here, we have assessed the effects of rimonabant in vitro in murine immortalized keratinocytes and in vivo by assaying the topical anti-inflammatory activity.

EXPERIMENTAL APPROACH

Cell viability and death in a keratinocyte cell line (C5N cells) were measured by Trypan blue exclusion assay and cytotoxicity by sulphorhodamine B test. Cell cycle progression was assessed by flow cytometry and the expression of apoptotic and anti-apoptotic markers, cyclins, pathways of signal transduction and CB1 receptor levels were evaluated by Western blot. The topical anti-inflammatory properties of rimonabant were analysed by inhibition of croton oil-induced ear dermatitis in mice.

KEY RESULTS

Rimonabant reduced cell viability and induced apoptosis as shown by the enhanced number of cells in the subG0 phase of the cell cycle, the expression of Bax and reduced levels of Bcl-2 and X-inhibitor of apoptosis protein. In addition, reduced levels of phosphorylated serine/threonine protein kinase Akt and nuclear factor-kappa B were detected associated with regulation of total nuclear factor-kappa B and inhibitor of kappa B-α, phosphorylated inhibitor of kappa B-α, cyclins D1, E and A. In croton oil-induced ear dermatitis, rimonabant significantly reduced oedema and leukocyte infiltrate.

CONCLUSIONS AND IMPLICATIONS

Rimonabant reduced cell viability, inducing cell death in keratinocytes and decreased croton oil-induced ear dermatitis. Our findings suggest a potential application of rimonabant as a topical anti-inflammatory drug. We did not assess the involvement of CB₁ receptors in these effects of rimonabant.     

Fasitibant chloride, a kinin B₂ receptor antagonist, and dexamethasone interact to inhibit carrageenan-induced inflammatory arthritis in rats

BACKGROUND AND PURPOSE

Bradykinin, through the kinin B₂ receptor, is involved in inflammatory processes related to arthropathies. B₂ receptor antagonists inhibited carrageenan-induced arthritis in rats in synergy with anti-inflammatory steroids. The mechanism(s) underlying this drug interaction was investigated.

EXPERIMENTAL APPROACH

Drugs inhibiting inflammatory mediators released by carrageenan were injected, alone or in combination, into the knee joint of pentobarbital anaesthetized rats 30 min before intra-articular administration of carrageenan. Their effects on the carrageenan-induced inflammatory responses (joint pain, oedema and neutrophil recruitment) and release of inflammatory mediators (prostaglandins, IL-1β, IL-6 and the chemokine GRO/CINC-1), were assessed after 6 h.

KEY RESULTS

The combination of fasitibant chloride (MEN16132) and dexamethasone was more effective than each drug administered alone in inhibiting knee joint inflammation and release of inflammatory mediators. Fasitibant chloride, MK571, atenolol, des-Arg⁹-[Leu⁸]-bradykinin (B₂ receptor, leukotriene, catecholamine and B₁ receptor antagonists, respectively) and dexketoprofen (COX inhibitor), reduced joint pain and, except for the latter, also diminished joint oedema. A combination of drugs inhibiting joint pain (fasitibant chloride, des-Arg⁹-[Leu⁸]-bradykinin, dexketoprofen, MK571 and atenolol) and oedema (fasitibant chloride, des-Arg⁹-[Leu⁸]-bradykinin, MK571 and atenolol) abolished the respective inflammatory response, producing inhibition comparable with that achieved with the combination of fasitibant chloride and dexamethasone. MK571 alone was able to block neutrophil recruitment.

CONCLUSIONS AND IMPLICATIONS

Bradykinin-mediated inflammatory responses to intra-articular carrageenan were not controlled by steroids, which were not capable of preventing bradykinin effects either by direct activation of the B₂ receptor, or through the indirect effects mediated by release of eicosanoids and cytokines.     

Endothelin-1 and endothelin-2 initiate and maintain contractile responses by different mechanisms in rat mesenteric and cerebral arteries

BACKGROUND AND PURPOSE

Endothelin (ET)-1 and ET-2 cause potent long-lasting vasoconstrictions by tight binding to smooth muscle ET_A receptors. We tested the hypotheses that different mechanisms mediate initiation and maintenance of arterial contractile responses to ET-1 and ET-2 and that this differs among vascular beds.

EXPERIMENTAL APPROACH

Segments of rat mesenteric resistance artery (MRA) and basilar artery (BA) were studied in wire myographs with and without functional antagonists.

KEY RESULTS

Sensitivity and maximum of MRA contractile responses to ET-1 were not, or only moderately, reduced by stimulation of soluble GC, AC or K⁺-channels and by an inhibitor of receptor-operated ion channels. However, each of these reduced maintenance of ET-1 effects and relaxed ET-1-induced contractions in MRA. A calcium channel antagonist did not alter sensitivity, maximum and maintenance of ET-1 effects, but relaxed ET-1-induced contractions in MRA. A PLC inhibitor prevented contractile responses to ET-1 and ET-2 in MRA and BA, and relaxed ET-1- and ET-2-induced responses in MRA and ET-1 effects in BA. A Rho-kinase inhibitor did not modify sensitivity, maximum and maintenance of responses to both peptides in both arteries but relaxed ET-2, but not ET-1, effects in MRA and ET-1 effects in BA.

CONCLUSIONS AND IMPLICATIONS

PLC played a key role in arterial contractile responses to ETs, but ET-1 and ET-2 initiated and maintained vasoconstriction through different mechanisms, and these differed between MRA and BA. Selective functional antagonism may be considered for agonist- and vascular bed selective pharmacotherapy of ET-related diseases.     

PPARα mediates the anti-inflammatory effect of simvastatin in an experimental model of zymosan-induced multiple organ failure

BACKGROUND AND PURPOSE

Zymosan-induced non-septic shock is a multi-factorial pathology that involves several organs including the kidneys, liver and lungs. Its complexity and diversity presents a continuing therapeutic challenge. Given their pleiotropic effect, statins could be beneficial in non-septic shock. One of the molecular mechanisms underlying the anti-inflammatory effect of statins involves the peroxisome proliferator-activated receptor (PPAR) α. We used a zymosan-induced non-septic shock experimental model to investigate the role of PPARα in the anti-inflammatory effects of simvastatin.

EXPERIMENTAL APPROACH

Effects of simvastatin (5 or 10 mg·kg⁻¹ i.p.) were analysed in PPARα knock-out (KO) and PPARα wild type (WT) mice after zymosan or vehicle administration. Organ injury in lung, liver, kidney and intestine was evaluated by immunohistology. PPARα mRNA expression and nuclear factor-κB activation were evaluated in all experimental groups, 18 h after study onset. Cytokine levels were measured in plasma, and nitrite/nitrate in plasma and peritoneal exudate. Nitric oxide synthase, nitrotyrosine and poly ADP-ribose were localized by immunohistochemical methods.

KEY RESULTS

Simvastatin significantly and dose-dependently increased the zymosan-induced expression of PPARα levels in all tissues analysed. It also dose-dependently reduced systemic inflammation and the organ injury induced by zymosan in lung, liver, intestine and kidney. These effects were observed in PPARαWT mice and in PPARαKO mice.

CONCLUSIONS AND IMPLICATIONS

Simvastatin protected against the molecular and cellular damage caused by systemic inflammation in our experimental model. Our results also provide new information regarding the role of PPARα in the anti-inflammatory effects of statins.     

Dual bronchodilatory and pulmonary anti-inflammatory activity of RO5024118, a novel agonist at vasoactive intestinal peptide VPAC2 receptors

BACKGROUND AND PURPOSE

Vasoactive intestinal peptide is expressed in the respiratory tract and induces its effects via its receptors, VPAC₁ and VPAC₂. RO5024118 is a selective VPAC₂ receptor agonist derived via chemical modification of an earlier VPAC₂ agonist, RO0251553. In the present studies, we characterized the pharmacological activity of RO5024118.

EXPERIMENTAL APPROACH

Stability of RO5024118 to human neutrophil elastase was assessed. Bronchodilatory activity of RO5024118 was investigated in guinea pig and human isolated airway smooth muscle preparations and in a guinea pig bronchoconstriction model. Pulmonary anti-inflammatory activity of RO5024118 was investigated in a lipopolysaccharide mouse model and in a porcine pancreatic elastase (PPE) rat model.

KEY RESULTS

RO5024118 demonstrated increased stability to neutrophil elastase compared with RO0251553. In human and guinea pig isolated airway preparations, RO5024118 induced bronchodilatory effects comparable with RO0251553 and the long-acting β-agonist salmeterol and was significantly more potent than native vasoactive intestinal peptide and the short-acting β-agonist salbutamol. In 5-HT-induced bronchoconstriction in guinea pigs, RO5024118 exhibited inhibitory activity with similar efficacy as, and longer duration than, RO0251553. In a lipopolysaccharide-mouse model, RO5024118 inhibited neutrophil and CD8⁺ cells and myeloperoxidase levels. In rats, intratracheal instillation of PPE induced airway neutrophilia that was resistant to dexamethasone. Pretreatment with RO5024118 significantly inhibited PPE-induced neutrophil accumulation.

CONCLUSIONS AND IMPLICATIONS

These results demonstrate that RO5024118 induces dual bronchodilatory and pulmonary anti-inflammatory activity and may be beneficial in treating airway obstructive and inflammatory diseases.     

Role of endothelin-1 in the hyper-responsiveness to nitrovasodilators following acute NOS inhibition

BACKGROUND AND PURPOSE

Acute NOS inhibition in humans and animals is associated with hypersensitivity to NO donors. The mechanisms underlying this phenomenon have not been fully elucidated. The purpose of the present study was to assess whether hypersensitivity to NOS-blockade is linked to endothelin-1 (ET-1) signalling.

EXPERIMENTAL APPROACH

Sprague Dawley rats were instrumented with indwelling arterial and venous catheters for continuous assessments of haemodynamic parameters and drug delivery, respectively. Mesenteric arteries were isolated and tested for reactivity by wire myography.

KEY RESULTS

NOS blockade with L-N^G-nitroarginine methyl ester (L-NAME) caused a pronounced increase in arterial blood pressure (BP) (∼40 mmHg). In L-NAME-treated animals, the dose of sodium nitroprusside (SNP) required to cause a significant reduction in arterial BP was lower than in vehicle-treated rats (P < 0.001), and the magnitude of the reduction in BP was greater. Similar results were obtained with other NO mimetics, but not isoprenaline; moreover, decreasing the BP back to baseline levels with prazosin after L-NAME treatment did not attenuate the hyper-responsiveness to NO donors. The increased responsiveness to NO donors was abolished by pretreatment with the ET_A/B receptor antagonist, PD145065, or the ET_A receptor-specific antagonist ABT627. Ex vivo, L-NAME treatment potentiated the constriction induced by big endothelin-1 (bET-1), the precursor to active ET-1, but had no effect on the ET-1-mediated constriction.

CONCLUSIONS AND IMPLICATIONS

These data suggest that the increased sensitivity to NO donors is mediated, at least in part, by ET-1 in vivo, and the mechanism may involve the conversion of bET-1 to ET-1.     

Maresin 1 mitigates LPS-induced acute lung injury in mice

BACKGROUND AND PURPOSE

Acute lung injury (ALI) is a severe illness with a high rate of mortality. Maresin 1 (MaR1) was recently reported to regulate inflammatory responses. We used a LPS-induced ALI model to determine whether MaR1 can mitigate lung injury.

EXPERIMENTAL APPROACH

Male BALB/c mice were injected, intratracheally, with either LPS (3 mg·kg⁻¹) or normal saline (1.5 mL·kg⁻¹). After this, normal saline, a low dose of MaR1 (0.1 ng per mouse) or a high dose of MaR1 (1 ng per mouse) was given i.v. Lung injury was evaluated by detecting arterial blood gas, pathohistological examination, pulmonary oedema, inflammatory cell infiltration, inflammatory cytokines in the bronchoalveolar lavage fluid and neutrophil–platelet interactions.

KEY RESULTS

The high dose of MaR1 significantly inhibited LPS-induced ALI by restoring oxygenation, attenuating pulmonary oedema and mitigating pathohistological changes. A combination of elisa and immunohistochemistry showed that high-dose MaR1 attenuated LPS-induced increases in pro-inflammatory cytokines (TNF-α, IL-1β and IL-6), chemokines [keratinocyte chemokine, monocyte chemoattractant protein-5, macrophage inflammatory protein (MIP)-1α and MIP-1γ], pulmonary myeloperoxidase activity and neutrophil infiltration in the lung tissues. Consistent with these observations, flow cytometry and Western blotting indicated that MaR1 down-regulated LPS-induced neutrophil adhesions and suppressed the expression of intercellular adhesion molecule (ICAM)-1, P-selection and CD24.

CONCLUSIONS AND IMPLICATIONS

High-dose MaR1 mitigated LPS-induced lung injury in mice by inhibiting neutrophil adhesions and decreasing the levels of pro-inflammatory cytokines.     

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.     

Increased endothelin-1 vasoconstriction in mesenteric resistance arteries after superior mesenteric ischaemia-reperfusion

BACKGROUND AND PURPOSE

Endothelin-1 (ET-1) plays an important role in the maintenance of vascular tone. We aimed to evaluate the influence of superior mesenteric artery (SMA) ischaemia-reperfusion (I/R) on mesenteric resistance artery vasomotor function and the mechanism involved in the changes in vascular responses to ET-1.

EXPERIMENTAL APPROACH

SMA from male Sprague-Dawley rats was occluded (90 min) and following reperfusion (24 h), mesenteric resistance arteries were dissected. Vascular reactivity was studied using wire myography. Protein and mRNA expression, superoxide anion (O₂^•−) production and ET-1 plasma concentration were evaluated by immunofluorescence, real-time quantitative PCR, ethidium fluorescence and elisa, respectively.

KEY RESULTS

I/R increased ET-1 plasma concentration, ET-1-mediated vasoconstriction and ET_B mRNA expression, and down-regulated ET_A mRNA expression. Immunofluorescence confirmed mRNA results and revealed an increase in ET_B receptors in the mesenteric resistance artery media layer after I/R. Therefore, the ET_B receptor agonist sarafotoxin-6 induced a contraction that was inhibited by the ET_B receptor antagonist BQ788 only in vessels, with and without endothelium, from I/R rats. Furthermore, BQ788 potentiated ET-1 vasoconstriction only in sham rats. Endothelium removal in rings from I/R rats unmasked the inhibition of ET-1 vasoconstriction by BQ788. Endothelium removal, N^ω-nitro-L-arginine methyl ester and superoxide dismutase abolished the differences in ET-1 vasoconstriction between sham and I/R rats. We also found that I/R down-regulates endothelial NOS mRNA expression and concomitantly enhanced O₂^•− production by increasing NADPH oxidase 1 (NOX-1) and p^47phox mRNA.

CONCLUSIONS AND IMPLICATIONS

Mesenteric I/R potentiated the ET-1-mediated vasoconstriction by a mechanism that involves up-regulation of muscular ET_B receptors and decrease in NO bioavailability.     

Neutrophil migration towards C5a and CXCL8 is prevented by non-steroidal anti-inflammatory drugs via inhibition of different pathways

BACKGROUND AND PURPOSE

Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to induce PG-independent anti-inflammatory actions. Here, we investigated the role of three different NSAIDs (naproxen, ibuprofen and oxaprozin) on neutrophil responses to CXCL8 and C5a.

EXPERIMENTAL APPROACH

Human neutrophils were isolated from healthy volunteers by dextran and Ficoll-Hypaque density gradients. Neutrophils were pre-incubated with different concentrations (1–100 µM) of NSAIDs or kinase inhibitors. Neutrophil degranulation into supernatants was tested by elisa and zymography. Neutrophil chemotaxis was determined using Boyden chambers. F-actin polymerization was determined by Alexa-Fluor 488-conjugated phalloidin fluorescent assay. Integrin expression was assessed by flow cytometry. The phosphorylation of intracellular kinases was studied by Western blot.

KEY RESULTS

Pretreatment with NSAIDs did not affect neutrophil degranulation, but inhibited neutrophil migration and polymerization of F-actin, in response to CXCL8 and C5a. Pretreatment with different NSAIDs prevented C5a-induced integrin (CD11b) up-regulation, while only ibuprofen reduced CXCL8-induced CD11b up-regulation. Pre-incubation with naproxen or oxaprozin, but not ibuprofen, inhibited the PI3K/Akt-dependent chemotactic pathways. Both endogenous (released in cell supernatants) or exogenous (added to cell cultures) PGE₂ did not affect C5a- or CXCL8-induced activities. Short-term incubation with NSAIDs did not affect neutrophil PGE₂ release.

CONCLUSION AND IMPLICATIONS

Treatment with NSAIDs reduced C5a- and CXCL8-induced neutrophil migration and F-actin polymerization via different mechanisms. Inhibition by ibuprofen was associated with integrin down-regulation, while naproxen and oxaprozin blocked the PI3K/Akt pathway. Both NSAID actions were independent of COX inhibition and PGE₂ release.     

TASK1 (K(2P)3.1) K(+) channel inhibition by endothelin-1 is mediated through Rho kinase-dependent phosphorylation

BACKGROUND AND PURPOSE

TASK1 (K_2P3.1) two-pore-domain K⁺ channels contribute substantially to the resting membrane potential in human pulmonary artery smooth muscle cells (hPASMC), modulating vascular tone and diameter. The endothelin-1 (ET-1) pathway mediates vasoconstriction and is an established target of pulmonary arterial hypertension (PAH) therapy. ET-1-mediated inhibition of TASK1 currents in hPASMC is implicated in the pathophysiology of PAH. This study was designed to elucidate molecular mechanisms underlying inhibition of TASK1 channels by ET-1.

EXPERIMENTAL APPROACH

Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record TASK1 currents from hPASMC and Xenopus oocytes.

KEY RESULTS

ET-1 inhibited TASK1-mediated I_KN currents in hPASMC, an effect attenuated by Rho kinase inhibition with Y-27632. In Xenopus oocytes, TASK1 current reduction by ET-1 was mediated by endothelin receptors ET_A (IC₅₀= 0.08 nM) and ET_B (IC₅₀= 0.23 nM) via Rho kinase signalling. TASK1 channels contain two putative Rho kinase phosphorylation sites, Ser³³⁶ and Ser³⁹³. Mutation of Ser³⁹³ rendered TASK1 channels insensitive to ET_A- or ET_B-mediated current inhibition. In contrast, removal of Ser³³⁶ selectively attenuated ET_A-dependent TASK1 regulation without affecting the ET_B pathway.

CONCLUSIONS AND IMPLICATIONS

ET-1 regulated vascular TASK1 currents through ET_A and ET_B receptors mediated by downstream activation of Rho kinase and direct channel phosphorylation. The Rho kinase pathway in PASMC may provide a more specific therapeutic target in pulmonary arterial hypertension treatment.     

Differing effects of exogenous and endogenous hydrogen sulphide in carrageenan-induced knee joint synovitis in the rat

Background and purpose:

Recent findings suggest that the noxious gas H₂S is produced endogenously, and that physiological concentrations of H₂S are able to modulate pain and inflammation in rodents. This study was undertaken to evaluate the ability of endogenous and exogenous H₂S to modulate carrageenan-induced synovitis in the rat knee.

Experimental approach:

Synovitis was induced in Wistar rats by intra-articular injection of carrageenan into the knee joint. Sixty minutes prior to carrageenan injection, the rats were pretreated with indomethacin, an inhibitor of H₂S formation (dl-propargylglycine) or an H₂S donor [Lawesson''s reagent (LR)].

Key results:

Injection of carrageenan evoked knee inflammation, pain as characterized by impaired gait, secondary tactile allodynia of the ipsilateral hindpaw, joint swelling, histological changes, inflammatory cell infiltration, increased synovial myeloperoxidase, protein nitrotyrosine residues, inducible NOS (iNOS) activity and NO production. Pretreatment with LR or indomethacin significantly attenuated the pain responses, and all the inflammatory and biochemical changes, except for the increased iNOS activity, NO production and 3-NT. Propargylglycine pretreatment potentiated synovial iNOS activity (and NO production), and enhanced macrophage infiltration, but had no effect on other inflammatory parameters.

Conclusions and implications:

Whereas exogenous H₂S delivered to the knee joint can produce a significant anti-inflammatory and anti-nociceptive effect, locally produced H₂S exerts little immunomodulatory effect. These data further support the development and use of H₂S donors as potential alternatives (or complementary therapies) to the available anti-inflammatory compounds used for treatment of joint inflammation or relief of its symptoms.