Acrolein increases 5-lipoxygenase expression in murine macrophages through activation of ERK pathway

Episodic exposure to acrolein-rich pollutants has been linked to acute myocardial infarction, and 5-lipoxygenase (5-LO) is involved in the production of matrix metalloproteinase-9 (MMP-9), which destabilizes atherosclerotic plaques. Thus, the present study determined the effect of acrolein on 5-LO/leukotriene B₄ (LTB₄) production in murine macrophages. Stimulation of J774A.1 cells with acrolein led to increased LTB₄ production in association with increased 5-LO expression. Acrolein-evoked 5-LO expression was blocked by pharmacological inhibition of the ERK pathway, but not by inhibitors for JNK and p38 MAPK pathways. In line with these results, acrolein exclusively increased the phosphorylation of ERK among these MAPK, suggesting a role for the ERK pathway in acrolein-induced 5-LO expression with subsequent production of LTB₄. Among the receptor tyrosine kinases including epidermal growth factor receptor (EGFR) and platelet derived growth factor receptor (PDGFR), acrolein-evoked ERK phosphorylation was attenuated by AG1478, an EGFR inhibitor, but not by AG1295, a PDGFR inhibitor. In addition, acrolein-evoked 5-LO expression was also inhibited by inhibition of EGFR pathway, but not by inhibition of PDGFR pathway. These observations suggest that acrolein has a profound effect on the 5-LO pathway via an EGFR-mediated activation of ERK pathway, leading to acute ischemic syndromes through the generation of LTB₄, subsequent MMP-9 production and plaque rupture.     

Augmentation of human neutrophil and alveolar macrophage LTB4 production by N-acetylcysteine: role of hydrogen peroxide

1. The actions of N-acetylcysteine (NAC) on hydrogen peroxide (H₂O₂) and leukotriene B₄ (LTB₄) production by human resting and stimulated peripheral blood neutrophils and alveolar macrophages were investigated.
2. At a concentration of 100 μM, NAC significantly (P<0.01) suppressed the accumulation of H₂O₂ in the incubation medium of resting and opsonized zymosan (OZ; 0.5 mg ml⁻¹)- or N-formylmethionyl-leucyl-phenylalanine (fMLP; 1 μM)-stimulated neutrophils and of resting and OZ-stimulated macrophages. At concentrations of 10 μM and above, NAC augmented significantly the level of LTB₄ in the supernatants of OZ- and fMLP-stimulated neutrophils (P<0.01 and P<0.05, respectively) and OZ-stimulated macrophages (P<0.05 at 10 μM, P<0.01 at 100 μM NAC).
3. NAC (100 μM) caused a significant (P<0.01) reduction in the quantity of measurable H₂O₂ when incubated with exogenous H₂O₂ concentrations equivalent to those released from OZ-stimulated neutrophils and macrophages. At no concentration did NAC affect quantitites of measurable LTB₄ when incubated with exogenous LTB₄.
4. Superoxide dismutase (SOD), which catalyzes the conversion of superoxide anion to H₂O₂ had no significant effect on LTB₄ production by human neutrophils. In contrast, catalase, which catalyzes the conversion of H₂O₂ to H₂O and O₂, caused a pronounced, statistically significant (P<0.01) increase in the levels of LTB₄ measured in the supernatants of OZ- and fMLP-stimulated neutrophils.
5. H₂O₂ (12.5 μM and 25 μM, concentrations equivalent to those measured in the supernatants of activated neutrophils and alveolar macrophages, respectively) caused a small (13%) decrease in the quantity of measurable LTB₄ (P=0.051 and P<0.05 at 12.5 μM and 25 μM, respectively) that was inhibited by NAC (100 μM) but not by catalase (400 u ml⁻¹).
6. In conclusion, the anti-oxidant drug, NAC, increases LTB₄ production by human neutrophils and alveolar macrophages, probably through the elimination of cell-derived H₂O₂. LTB₄ undergoes a H₂O₂-dependent oxidation that is inhibited by NAC but this is unlikely to account fully for the increased levels of LTB₄, suggesting that NAC may increase LTB₄ production by blocking the H₂O₂-dependent inhibition of a synthetic enzyme, such as 5-lipoxygenase.

     

Mechanisms of the influence of magnolol on eicosanoid metabolism in neutrophils

We have demonstrated that magnolol suppressed thromboxane B2 (TXB2) and leukotriene B4 (LTB4) formation in A23187-stimulated rat neutrophils. Maximum inhibition was obtained with about 10 microM magnolol. Magnolol was more effective in the inhibition of cyclooxygenase (COX) activity than in the inhibition of 5-lipoxygenase (5-LO) activity as assessed by means of enzyme activity determination in vitro and COX and 5-LO metabolic capacity analyses in vivo. Magnolol alone stimulated cytosolic phospholipase A2 (cPLA2) phosphorylation and the translocation of 5-LO and cPLA2 to the membrane, and evoked arachidonic acid (AA) release. Recruitment of both 5-LO and cPLA2 to the membranes was suppressed by EGTA. Arachidonyl trifluoromethyl ketone (AACOCF3), a PLA2 inhibitor, bromoenol lactone (BEL), a Ca2+-independent PLA2 (iPLA2) inhibitor, and EGTA suppressed the magnolol-induced AA release. However, none of the follows affected magnolol-induced AA-release: 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), a p38 mitogen-activated protein kinase (MAPK) inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), a MAPK kinase (MEK) inhibitor, or 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF109203X), a protein kinase C (PKC) inhibitor. In addition, magnolol at 30 microM did not stimulate the p38 MAPK and extracellular signal-regulated kinase 2 (ERK2) enzyme activities. These results indicated that magnolol inhibits the formation of prostaglandins and leukotrienes in A23187-stimulated rat neutrophils, probably through a direct blockade of COX and 5-LO activities. The stimulatory effects of magnolol at high concentration on the membrane association of 5-LO and cPLA2 are attributable to the elevation of [Ca2+]i, and on the AA release is likely via activation of cPLA2 and iPLA2.     

Leukotriene B4 receptor antagonists as therapeutics for inflammatory disease: preclinical and clinical developments

Leukotriene B₄ (LTB₄) is a lipid inflammatory mediator derived from membrane phospholipids by the sequential actions of cytosolic phospholipase A2 (PLA2), 5-lipoxygenase (5-LO) and leukotriene A₄ (LTA₄) hydrolase. Several inflammatory diseases, including asthma, chronic obstructive pulmonary disease, arthritis and inflammatory bowel disease, have been associated with elevated levels of LTB₄. As a result, pharmacological strategies to modulate the synthesis of LTB₄ (inhibition of PLA2, 5-LO or LTA₄ hydrolase) or the effects of LTB₄ itself (antagonism of LTB₄ receptors) are being developed by several companies. Two G-protein-coupled receptors mediate the effects of LTB₄, namely BLT1 and BLT2. The pharmacology, expression and function of these two receptors were last reviewed by Tager and Luster in 2004. Since then, there has been an increased understanding of the function of these receptors, in particular for the lesser understood of the two receptors, BLT2. Furthermore, since last reviewed in 1996, there have been several clinical developments in the use of BLT receptor antagonists for inflammatory diseases. This review summarizes the latest preclinical and clinical developments in BLT antagonism for inflammatory diseases and discusses potential future developments.     

Novel leukotriene biosynthesis inhibitors (2012-2016) as anti-inflammatory agents

Introduction: Leukotrienes (LTs) are lipid mediators produced from arachidonic acid with a broad variety of bioactivities in allergy and inflammation. The biosynthesis of LTs mainly involves 5-lipoxygenase (5-LO) and its 5-lipoxygenase-activating protein (FLAP), LTA₄ hydrolase and LTC₄ synthase that all may represent potential targets for LT biosynthesis inhibitors.

Areas covered: We introduce the LT biosynthetic pathway and its cellular regulation, the diverse biological actions of LTs and their receptors, and we briefly describe the pharmacological strategies for suppression of LT formation as well as the classes of current LT biosynthesis inhibitors. The main focus is placed on the comprehensive discussion of recently reported inhibitors of 5-LO, FLAP, LTA₄ hydrolase and LTC₄ synthase, based on literature search (PubMed and Thomson Innovation Patents Searches), covering 2012–2016.

Expert opinion: Although many new series of 5-LO inhibitors have been presented without patenting, essentially by academia, novel FLAP inhibitors (many patented) are most advanced in clinical development and are apparently the focus of pharmaceutical companies. Only few novel inhibitors of LTA₄ hydrolase and LTC₄ synthase were reported. Major issues in the development of LT synthesis inhibitors are related to loss of potency in biological relevant environment, poor pharmacokinetics, lack of oral efficacy, and side effects.     

Saucerneol F,a New Lignan Isolated from Saururus chinensis,Attenuates Degranulation via Phospholipase Cγ 1 Inhibition and Eicosanoid Generation by Suppressing MAP Kinases in Mast Cells

During our on-going studies to identify bioactive compounds in medicinal herbs, we found that saucerneol F (SF), a naturally occurring sesquilignan isolated from Saururus chinensis (S. chinensis), showed in vitro anti-inflammatory activity. In this study, we examined the effects of SF on the generation of 5-lipoxygenase (5-LO) dependent leukotriene C₄ (LTC₄), cyclooxygenase-2 (COX-2) dependent prostaglandin D₂ (PGD₂), and on phospholipase Cγ1 (PLCγ1)-mediated degranulation in SCF-induced mouse bone marrow-derived mast cells (BMMCs). SF inhibited eicosanoid (PGD₂ and LTC₄) generation and degranulation dose-dependently. To identify the molecular mechanisms underlying the inhibition of eicosanoid generation and degranulation by SF, we examined the effects of SF on the phosphorylation of PLCγ1, intracellular Ca²⁺ influx, the translocation of cytosolic phospholipase A₂ (cPLA₂) and 5-LO, and on the phosphorylation of MAP kinases (MAPKs). SF was found to reduce intracellular Ca²⁺ influx by inhibiting PLCγ1 phosphorylation and suppressing the nuclear translocations of cPLA₂ and 5-LO via the phosphorylations of MAPKs, including extracellular signal-regulated protein kinase-1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. Taken together, these results suggest that SF may be useful for regulating mast cell-mediated inflammatory responses by inhibiting degranulation and eicosanoid generation.     

On the inhibition of 5-lipoxygenase product formation by tryptanthrin: mechanistic studies and efficacy in vivo

BACKGROUND AND PURPOSE

Leukotrienes (LTs) are pro-inflammatory mediators produced by 5-lipoxygenase (5-LO). Currently available 5-LO inhibitors either lack efficacy or are toxic and novel approaches are required to establish a successful anti-LT therapy. Here we provide a detailed evaluation of the effectiveness of the plant-derived alkaloid tryptanthrin as an inhibitor of LT biosynthesis.

EXPERIMENTAL APPROACH

We analysed LT formation and performed mechanistic studies in human neutrophils stimulated with pathophysiologically relevant stimuli (LPS and formyl peptide), as well as in cell-free assays (neutrophil homogenates or recombinant human 5-LO) and in human whole blood. The in vivo effectiveness of tryptanthrin was evaluated in the rat model of carrageenan-induced pleurisy.

KEY RESULTS

Tryptanthrin potently reduced LT-formation in human neutrophils (IC₅₀ = 0.6 µM). However, tryptanthrin is not a redox-active compound and did not directly interfere with 5-LO activity in cell-free assays. Similarly, tryptanthrin did not inhibit the release of arachidonic acid, the activation of MAPKs, or the increase in [Ca²⁺]_i, but it modified the subcellular localization of 5-LO. Moreover, tryptanthrin potently suppressed LT formation in human whole blood (IC₅₀ = 10 µM) and reduced LTB₄ levels in the rat pleurisy model after a single oral dose of 10 mg·kg⁻¹.

CONCLUSIONS AND IMPLICATIONS

Our data reveal that tryptanthrin is a potent natural inhibitor of cellular LT biosynthesis with proven efficacy in whole blood and is effective in vivo after oral administration. Its unique pharmacological profile supports further analysis to exploit its pharmacological potential.     

Development of 5-lipoxygenase inhibitors--lessons from cellular enzyme regulation

5-Lipoxygenase (5-LO) catalyzes the first steps in the conversion of arachidonic acid (AA) into leukotrienes (LTs) that are mediators of inflammatory and allergic reactions. Recently, the 5-LO pathway has also been associated with atherosclerosis and osteoporosis. Thus, in addition to the classical applications including asthma and allergic disorders, LT synthesis inhibitors might be of interest for the treatment of cardiovascular diseases and osteoporosis. Recently, it has been shown that cellular 5-LO activity is regulated in a complex manner that can involve different signalling pathways. 5-LO can be activated by an increase in intracellular Ca²⁺ concentration, diacylglycerols, phosphorylation by MAPKAP kinase-2 and ERK. Previous work could demonstrate that cellular 5-LO activity is repressed in a protein kinase A-dependent manner and by glutathione peroxidases. This comment focuses on the impact of these stimulatory and inhibitory pathways on the efficacy of 5-LO inhibitors and suggests additional criteria for the development of this class of compounds.     

Targeting leukotriene B4 in inflammation

Introduction: Leukotriene (LT) B₄ is a powerful proinflammatory lipid mediator and triggers adherence to the endothelium, activates and recruits leukocytes to the site of injury. When formed in excess, LTB₄ plays a pathogenic role and may sustain chronic inflammation in diseases such as asthma, rheumatoid arthritis, and inflammatory bowel disease. Recent investigations have also indicated that LTB₄ is involved in cardiovascular diseases.

Areas covered: As the 5-lipoxygenase pathway involves several discrete, tightly coupled, enzymes, which convert the substrate, ‘step by step', into bioactive products, several different strategies have been used to target LTB₄ as a means to treat inflammation. Here, we discuss recent findings regarding the development of selective enzyme inhibitors and antagonists for LTB₄ receptors, as well as their application in preclinical and clinical studies.

Expert opinion: Components of the 5-lipoxygenase pathway have received considerable attention as candidate drug targets resulting in one new class of medications against asthma, that is, the antileukotrienes. However, efforts to specifically target LTB₄ have not yet been fruitful in the clinical setting, in spite of very promising preclinical data. Recently, crystal structures along with hitherto unknown functions of key enzymes in the leukotriene cascade have emerged, offering new opportunities for drug development and, with time, pharmacological intervention in LTB₄-mediated pathologies.     

Saucerneol D inhibits eicosanoid generation and degranulation through suppression of Syk kinase in mast cells

Previously we reported that saucerneol D (SD), a naturally occurring sesquilignan isolated from Saururus chinensis (S. chinensis) suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cells. The aim of this study was to elucidate whether SD modulates the generation of other inflammatory mediators in activated mast cells. We investigated the effects of SD on cyclooxygenase-2 (COX-2)-dependent prostaglandin D₂ (PGD₂) and 5-lipoxygenase (5-LO)-dependent leukotriene C₄ (LTC₄) generations as well as degranulation in cytokine-stimulated mouse bone marrow-derived mast cells (BMMCs). Biochemical analyses of the cytokine-mediated signaling pathways showed that SD suppressed the phosphorylation of Syk kinase and multiple downstream signaling processes including phospholipase Cγ1 (PLCγ1)-mediated intracellular Ca²⁺ influx and activation of mitogen-activated protein kinases (MAPKs; including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH₂-terminal kinase (JNK) and p38) and the nuclear factor-κB (NF-κB) pathway. Taken together, the present study suggests that SD suppresses eicosanoid generation and degranulation through Syk-dependent pathway in BMMCs.     

Role of p38 mapk on the down-regulation of matrix metallo-proteinase-9 expression in rat astrocytes

In spite of their pathophysiological importance in neuro-inflammatory diseases, little is known about the signal transduction pathways that lead to the induction of matrix metalloproteinases (MMPs) in the central nervous system. We reported previously that lipopolysaccharide (LPS) induced MMP-9 expression through ERK1/2 pathway in rat primary astrocytes (Glia 41:15-24, 2003). Here, we investigated the role of other MAPK pathways, including p38 and JNK/SAPK, on the regulation of MMP-9 expression in LPS-stimulated rat primary astrocytes. LPS activated both p38 and JNK in astrocytes. Treatment with a specific p38 MAPK inhibitor SB203580, but not JNK inhibitor SP600125, increased the LPS-stimulated MMP-9 expression in a concentration-dependent manner. Anti-inflammatory cytokines, including IFN-gamma and IL-4, activated p38 MAPK and decreased MMP-9 production in LPS-stimulated astrocytes. When p38 MAPK activation was blocked by SB203580, the inhibitory effects of these cytokines on MMP-9 induction were abolished. Finally, direct injection of SB203580 into the lateral ventricle of rat brain increased the LPS-induced MMP-9 activity in cerebral cortex. Altogether, these results suggest that p38 activation down-regulates the inflammatory stimulation-induced overexpression of MMP-9, both in primary astrocytes and in cerebral cortex. The elaborate interplay between ERK1/2 and p38 pathways provides a more sophisticated mechanism for regulating MMP-9 activity in neuroinflammatory diseases.     

Current status of leukotriene A4 hydrolase inhibitors

Background: Leukotriene A₄ hydrolase (LTA₄H) is a ubiquitously distributed 69-kDa zinc-containing cytosolic enzyme with both hydrolase and aminopeptidase activity. As a hydrolase, LTA₄H stereospecifically catalyzes the transformation of the unstable epoxide LTA₄ to the diol leukotriene B₄ (LTB₄), a potent chemoattractant and activator of neutrophils and a chemoattractant of eosinophils, macrophages, mast cells, T cells, dendritic cells, smooth muscle cells and keratinocytes. Inhibiting the formation of LTB₄ is expected to be beneficial in the treatment of inflammatory diseases such as inflammatory bowel disease, asthma and atherosclerosis. Objective: The focus of this review will be on patent applications/patents containing LTA₄H inhibitors that were published between 1996 and March 2008 from four different companies. The documents reviewed are presented in a tabular manner. Method: The first part of this review focuses on the primary literature supporting LTA₄H as a potential target. The second part covers the patent literature organized by applicant identified during the prescribed period. Conclusion: Activity towards identifying small molecule inhibitors of LTA4H has escalated in the last few years in line with important scientific and clinical developments involving the leukotriene pathway. One inhibitor from deCODE, DG-051, is reported to be in clinical trials for myocardial infarction and future reports of clinical efficacy will undoubtedly stimulate further work in the field by others.     

The development of methodology for clinical measurement of 5-lipoxygenase pathway intermediates from human peripheral blood mononuclear cells

Recent studies have shown a correlation between 5-lipoxygenase (5-LO) pathway up-regulation and cardiovascular risk. Despite the existence of several assays for products of the 5-LO pathway, a reliable method for clinical determination of 5-LO activity remains to be established. In the present communication, we report conditions that allow measurement of 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B₄ (LTB₄) in peripheral blood mononuclear cells (PBMCs) isolated from the blood of atherosclerosis patients before and after stimulation by the calcium ionophore, A23187. LTB₄, a potent mediator of inflammation-linked cardiovascular disease, was measured using an existing competitive enzyme immunoassay (EIA) kit after making specific methodological improvements that allowed PBMCs to be used in this format for the first time. LTB₄ was also measured by LC/MS/MS along with 5-HETE, a direct by-product of the action of 5-LO on arachidonic acid and a molecule for which no commercial EIA kit exists. The LC/MS/MS assay was validated over a range of 0.025–25 ng/mL for LTB₄ and 0.1–25 ng/mL for 5-HETE. The EIA method has a validated range covering 0.025–4 ng/mL. When both assays were applied to analyze LTB₄ from stimulated PBMCs isolated from 25 subjects with various degrees of atherosclerosis, a high correlation was obtained (r = 0.9426, Pearson's correlation coefficient). A high correlation was also observed between the levels of LTB₄ and 5-HETE measured by LC/MS/MS after ionophore stimulation (r = 0.9159). Details are presented for optimized sample collection, processing, storage, and analysis in accordance with the logistical demands of clinical analysis.     

Silibinin Inhibits LPS-Induced Macrophage Activation by Blocking p38 MAPK in RAW 264.7 Cells

We demonstrate herein that silibinin, a polyphenolic flavonoid compound isolated from milk thistle (Silybum marianum), inhibits LPS-induced activation of macrophages and production of nitric oxide (NO) in RAW 264.7 cells. Western blot analysis showed silibinin inhibits iNOS gene expression. RT-PCR showed that silibinin inhibits iNOS, TNF-α, and IL1β. We also showed that silibinin strongly inhibits p38 MAPK phosphorylation, whereas the ERK1/2 and JNK pathways are not inhibited. The p38 MAPK inhibitor abrogated the LPS-induced nitrite production, whereas the MEK-1 inhibitor did not affect the nitrite production. A molecular modeling study proposed a binding pose for silibinin targeting the ATP binding site of p38 MAPK (1OUK). Collectively, this series of experiments indicates that silibinin inhibits macrophage activation by blocking p38 MAPK signaling.     

Involvement of p38 MAPK- and JNK-modulated expression of Bcl-2 and Bax in Naja nigricollis CMS-9-induced apoptosis of human leukemia K562 cells

CMS-9, a phospholipase A₂ (PLA₂) isolated from Naja nigricollis venom, induced apoptosis of human leukemia K562 cells, characterized by mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Moreover, an increase in intracellular Ca²⁺ concentration and the production of reactive oxygen species (ROS) was noted. Pretreatment with BAPTA-AM (Ca²⁺ chelator) and N-acetylcysteine (NAC, ROS scavenger) proved that Ca²⁺ was an upstream event in inducing ROS generation. Upon exposure to CMS-9, activation of p38 MAPK and JNK was observed in K562 cells. BAPTA-AM or NAC abrogated CMS-9-elicited p38 MAPK and JNK activation, and rescued viability of CMS-9-treated K562 cells. SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) suppressed CMS-9-induced dissipation of mitochondrial membrane potential, Bcl-2 down-regulation, Bax up-regulation and increased mitochondrial translocation of Bax. Inactivation of PLA₂ activity reduced drastically the cytotoxicity of CMS-9, and a combination of lysophosphatidylcholine and stearic acid mimicked the cytotoxic effects of CMS-9. Taken together, our data suggest that CMS-9-induced apoptosis of K562 cells is catalytic activity-dependent and is mediated through mitochondria-mediated death pathway triggered by Ca²⁺/ROS-evoked p38 MAPK and JNK activation.     

几种抗炎药对白三烯B4生物合成的影响

白三烯B₄为花生四烯酸5-脂氧酶代谢产物,是炎症反应中的重要介质。目前抑制白三烯B₄生物合成的药物尚不多见。本文建立了测定白细胞来源白三烯B₄的反相高效液相色谱法,并初步探讨了几种抗炎药对白三烯B₄生物合成的影响。结果表明阿斯匹林、消炎痛、炎痛喜康、氢化可的松及麝香第一色带对白三烯B₄生物合成几无影响,而氟灭酸可明显抑制白三烯B₄生成,提示它可能是5-脂氧酶抑制剂。     

p38 MAPK and MMP-9 cooperatively regulate mucus overproduction in mice exposed to acrolein fog

ObjectiveTo evaluate the role of p38 mitogen-activated protein kinase (MAPK) on mice airway inflammation, mucus production and the possible cross-talk between p38 MAPK and matrix metalloproteinase-9 (MMP-9) in mucin protein synthesis.MethodsMice were exposed to 4.0 ppm of acrolein for 21 days with daily intraperitoneal injection of SB203580, a specific inhibitor of p38 MAPK. In control mice, sterile saline was administered instead. On days 7 and 21, mice were sacrificed to examine airway inflammation and mucus production by BALF cell counts, cytokine ELISA, and H&;E and AB-PAS staining. The mRNA and protein levels of Muc5ac, p38 MAPK and MMP-9 in the lung were determined by RT-PCR, immunohistochemistry and Western blotting analysis. MMP-9 activity was measured by gelatin zymography.ResultsBoth the numbers of inflammatory cells and mucus-secreting goblet cells were significantly increased in the airways of mice exposed to acrolein as compared to the control mice. Acrolein-increased phosphorylation of p38 MAPK was significantly reduced by SB203580. The airway inflammation and goblet cell hyperplasia after acrolein challenge were also attenuated by SB203580 administration. Moreover, SB203580 treatment decreased the acrolein-induced increase of Muc5ac and MMP-9 expression and MMP-9 activity in airway epithelium.ConclusionsThe results indicate an important role of p38 MAPK in acrolein-induced airway inflammation and mucus hypersecretion in mice. The cooperation of p38 and MMP-9 may contribute to the mucin overproduction after inflammatory challenge.     

Dual effect of the novel peptide antagonist K-14585 on proteinase-activated receptor-2-mediated signalling

Background and purpose:

Here we have examined the effects of the novel peptide antagonist N-[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indol-5-yl]aminocarbonyl}-glycinyl-L-lysinyl-L-phenylalanyl-N-benzhydrylamide (K-14585) on proteinase-activated receptor (PAR)₂-mediated intracellular signalling events.

Experimental approach:

Using NCTC2544 cells expressing PAR₂, we assessed the effects of K-14585 on PAR₂-mediated [³H] inositol phosphate accumulation, MAP kinase activation, p65 NFκB phosphorylation and DNA binding and IL-8 production.

Key results:

Pretreatment with K-14585 (5 µM) inhibited [³H] inositol phosphate levels stimulated by PAR₂-activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV-OH) in PAR₂-expressing NCTC2544 cells. K-14585 pretreatment did not influence PAR₂-mediated extracellular regulated kinase activation but inhibited p38 MAP kinase phosphorylation. At a higher concentration (30 µM), K-14585 alone stimulated p38 MAP kinase activation. These effects were replicated in EAhy926 cells, endogenously expressing PAR₂, but not in parental or PAR₄-expressing NCTC2544 cells, suggesting these effects were PAR₂-dependent. SLIGKV-mediated stimulation of p38 MAP kinase phosphorylation was substantially reduced by the G_q/11 inhibitor YM-254890, without affecting K-14585-mediated phosphorylation. Pretreatment with K-14585 inhibited PAR₂-mediated p65 NFκB phosphorylation and NFκB-DNA binding. K-14585 (30 µM) alone stimulated comparable NFκB reporter activity to SLIGKV-OH. K-14585 inhibited SLIGKV-stimulated IL-8 production, but given alone increased IL-8. While SLIGKV-induced IL-8 formation was reduced by both SB203580 and YM-254890, the response to K-14585 was sensitive to SB203580 but not YM-254890.

Conclusions and implications:

These data reveal that K-14585 has a duality of action functioning both as an antagonist and agonist due to either partial agonist actions or possible agonist-directed signalling. The data also suggest two modes of p38 MAP kinase activation emanating from PAR₂, one G_q/11-dependent and the other G_q/11-independent.     

An underlying role for hepatobiliary dysfunction in cyclosporine A nephrotoxicity

Renal-derived cysteinyl leukotrienes (cysLT), such as leukotrienes C(4) (LTC(4)) and D(4) (LTD(4)) are thought to mediate acute and chronic cyclosporine A (CSA) nephrotoxicity. However, whole-body cysLT elimination is regulated primarily by hepatobiliary excretion. Since CSA is known to alter hepatobiliary function, the effects of CSA on whole-body cysLT elimination were investigated in vivo, with respect to hepatobiliary and renal function. Male rats were anesthetized and cannulated (jugular vein, bile duct, and urinary bladder). A tracer dose of tritiated LTC(4) ((3)H-LTC(4)) was administered systemically (i.v.) immediately following vehicle and then 90 min later after vehicle or CSA. In vehicle/vehicle controls, hepatobiliary (3)H-cysLT elimination predominated over renal elimination without altering glomerular filtration rate (GFR), bile flow, and urine production. (3)H-cysLT elimination kinetics were comparable between each 90 min collection period. In vehicle/CSA-treated rats, an acutely nephrotoxic dose of CSA (20 mg/kg, i.v.) reduced urine flow 74+/-9% and caused a transient reduction in GFR, while total bile flow decreased 40+/-13%. Hepatobiliary and renal (3)H-cysLT elimination was also impaired 59+/-5 and 61+/-18%, respectively. In contrast, a non-nephrotoxic dose (2 mg/kg i.v.) increased renal (3)H-cysLT elimination due to impaired hepatobiliary elimination without affecting GFR, bile flow or urine production. Both doses caused (3)H-cysLT retention in hepatic and renal tissue. These findings demonstrate that CSA alters whole-body handling of cysLT by disrupting hepatobiliary cysLT elimination. This disruption leads to increased renal exposure to systemically derived cysLT and renal cysLT tissue retention. Renal exposure to and accumulation of systemically derived cysLT products may be underlying factors in CSA nephrotoxicity.