Ramatroban Research Articles

Mechanism of thromboxane receptor-induced vasoconstriction in human saphenous vein

Saphenous vein (SV) is one of the most widely used graft material in patients undergoing coronary artery bypass graft surgery (CABG). Thromboxane A2 (TXA2) is implicated in graft failure by inducing vasoconstriction and platelet aggregation. The aim of this study is to investigate the mechanism involved in TXA2-induced vasoconstriction in human SV. The role of different inhibitors and blockers on U46619 (TXA2-mimetic)-induced vasoconstriction is investigated by using an isolated organ bath system. Relaxation responses to several mediators are evaluated in SV pre-contracted with U46619 and compared with those pre-contracted with phenylephrine. Our results demonstrate that U46619-induced contraction is completely blocked by myosin light chain kinase inhibitor ML-9 or TP receptor antagonist BAY u3405. Furthermore, U46619-induced contraction is partially inhibited by phospholipase C inhibitor U73122, protein kinase C inhibitor calphostin C, Rho-kinase inhibitor Y-27632, L-type calcium channel blocker nifedipine, store-operated channel inhibitor SKF96365 or removal of extracellular calcium. Relaxation responses to NO donor (sodium nitroprusside), guanylate cyclase (GC) stimulator (riociguat), phosphodiesterase (PDE) inhibitors (sildenafil, IBMX), adenylate cyclase (AC) activator (forskolin) and acetylcholine (ACh) are markedly reduced when U46619 is used as a pre-contraction agent. Our results demonstrate that influx of extracellular Ca2+ (through L-type calcium channels and store-operated calcium channels) and intracellular Ca2+ release together with Ca2+ sensitization (through Rho-kinase activation) are necessary components for TXA2-induced vasoconstriction in SV. Moreover, more pronounced decrease in vasorelaxation induced by several mediators (SNP, riociguat, sildenafil, IBMX, forskolin, and ACh) in the presence of U46619 when compared with phenylephrine suggests that there is a crosstalk between the TP receptor signaling pathway and PDE, AC, GC enzymes. We believe that the investigation of mechanism of the TXA2-induced vasoconstriction in SV will provide additional information for the prevention of SV graft failure.

Cysteinyl leukotriene receptor (CysLT) antagonists decrease pentylenetetrazol-induced seizures and blood–brain barrier dysfunction

Current evidence suggests that inflammation plays a role in the pathophysiology of seizures. In line with this view, selected pro-inflammatory arachidonic acid derivatives have been reported to facilitate seizures. Kainate-induced seizures are accompanied by leukotriene formation, and are reduced by inhibitors of LOX/COX pathway. Moreover, LTD4 receptor blockade and LTD4 synthesis inhibition suppress pentylenetetrazol (PTZ)-induced kindling and pilocarpine-induced recurrent seizures. Although there is convincing evidence supporting that blood–brain-barrier (BBB) dysfunction facilitates seizures, no study has investigated whether the anticonvulsant effect of montelukast is associated with its ability to maintain BBB integrity. In this study we investigated whether montelukast and other CysLT receptor antagonists decrease PTZ-induced seizures, as well as whether these antagonists preserve BBB during PTZ-induced seizures.Adult male albino Swiss mice were stereotaxically implanted with a cannula into the right lateral ventricle, and two electrodes were placed over the parietal cortex along with a ground lead positioned over the nasal sinus for electroencephalography (EEG) recording. The effects of montelukast (0.03 or 0.3μmol/1μL, i.c.v.), pranlukast (1 or 3μmol/1μL, i.c.v.), Bay u-9773 (0.3, 3 or 30nmol/1μL, i.c.v.), in the presence or absence of the agonist LTD4 (0.2, 2, 6 or 20pmol/1μL, i.c.v.), on PTZ (1.8μmol/2μL)-induced seizures and BBB permeability disruption were determined. The animals were injected with the antagonists, agonist or vehicle 30min before PTZ, and monitored for additional 30min for the appearance of seizures by electrographic and behavioral methods. BBB permeability was assessed by sodium fluorescein method and by confocal microscopy for CD45 and IgG immunoreactivity. Bay-u9973 (3 and 30nmol), montelukast (0.03 and 0.3μmol) and pranlukast (1 and 3μmol), increased the latency to generalized seizures and decreased the mean amplitude of EEG recordings during seizures. LTD4 (0.2 and 2pmol) reverted the anticonvulsant effect of montelukast (0.3μmol). Montelukast (0.03 and 0.3μmol) prevented PTZ-induced BBB disruption, an effect that was reversed by LTD4 at the dose of 6pmol, but not at the doses 0.2 and 2pmol. Moreover, the doses of LTD4 (0.2 and 2pmol) that reverted the effect of montelukast on seizures did not alter montelukast-induced protection of BBB, dissociating BBB protection and anticonvulsant activity. Confocal microscopy analysis revealed that 1. PTZ increased the number of CD45+ and double-immunofluorescence staining for CD45 and IgG cells in the cerebral cortex, indicating BBB leakage with leukocyte infiltration; 2. while LTD4 (6pmol) potentiated, montelukast decreased the effect of PTZ on leukocyte migration and BBB, assessed by double-immunofluorescence staining for CD45 and IgG cells in the cannulated hemisphere. Our data do not allow us ruling out that mechanisms unrelated and related to BBB protection may co-exist, resulting in decreased seizure susceptibility by montelukast. Notwithstanding, they suggest that CysLT1 receptors may be a suitable target for anticonvulsant development.

Leukotriene-induced contraction is mediated by cysteinyl leukotriene receptor CysLT1 in guinea pig fundus but by CysLT1 and CysLT2 in antrum

Aims Leukotriene D 4 (LTD 4) causes contraction of the stomach through unclear receptors. The aim of the present study is to characterize the cysteinyl leukotriene receptor (CysLT) mediating leukotriene-induced muscle contraction in the stomach. Main methods We measured contraction of gastric muscle strips isolated from the guinea pig fundus and antrum caused by cysteinyl leukotrienes, including LTC 4, LTD 4 and LTE 4, as well as the dihydroxy leukotriene LTB 4 in vitro. Key findings In both fundic and antral muscle strips, LTC 4 and LTD 4 caused marked whereas LTE 4 caused moderate, concentration-dependent contractions. In contrast, LTB 4 caused only small contraction. The relative potencies for cysteinyl leukotrienes to cause contraction in both fundus and antrum were LTC 4 = LTD 4 > LTE 4. The LTD 4-induced contraction was not affected by tetrodotoxin or atropine, suggesting that the action is not neurally mediated. The LTD 4-induced contraction in the fundus was almost abolished by the CysLT 1 selective antagonist montelukast. In contrast, the LTD 4-induced contraction in the antrum was only partially inhibited by montelukast or the dual CysLT 1 and CysLT 2 antagonist BAY u9773. This antral contraction was almost abolished by the combination of montelukast and BAY u9773, indicating enhancement of inhibition. Significance The results of the present study demonstrate that cysteinyl leukotrienes LTC 4, LTD 4 and LTE 4 cause moderate to marked whereas the dihydroxy leukotriene LTB 4 causes small muscle contraction in the stomach in vitro. The leukotriene-induced contraction is mediated by CysLT 1 in fundus but by CysLT 1 and CysLT 2 in antrum.

Involvement of prostaglandin F2α in preeclamptic human umbilical vein vasospasm: a role of prostaglandin F and thromboxane A2 receptors

Preeclampsia is characterized by hypertension and proteinuria developing after 20 weeks of gestation. Increased vasoconstriction can be one of the major underlying pathophysiological event in this syndrome. We examined the role of vasoconstrictor prostanoid, prostaglandin F2α (PGF2α) in preeclamptic and normotensive human umbilical veins. Umbilical veins were set up in organ bath. The concentration-response curves of PGF2α (endogenous agonist of prostaglandin F receptor) and fluprostenol (prostaglandin F receptor selective agonist) were determined in normal and preeclamptic veins either in the absence or presence of BAY u3405 (thromboxane A2 receptor selective antagonist). PGF2α and its major metabolite concentrations were measured by enzyme immunoassay kit. The expression of vasoconstrictor prostanoid receptors was determined by western blot. The concentration-dependent contractions to PGF2α and fluprostenol were significantly increased in umbilical vein preparations derived from preeclamptic women compared with those of normotensives. Increased reactivity was related with enhanced sensitivity to these spasmogens in preeclamptic veins. BAY u3405 (10 μmol/l) did not modify the responsiveness to PGF2α in normal umbilical veins whereas moderately reduced the contractions in preeclamptic preparations. Serum concentrations of PGF2α and its major metabolite, 13,14-dihydro-15-keto-PGF2α, were comparable between preeclamptics and normotensives whereas the metabolite concentration was elevated in umbilical cord serum of preeclamptics. 13,14-dihydro-15-keto-PGF2α, release was also increased in umbilical vein preparations of preeclamptic women. An increased prostaglandin F receptor protein expression was determined whereas EP3 and thromboxane A2 protein expressions were unchanged in preeclamptic umbilical veins. Prostaglandin F and thromboxane A2 receptors activation by PGF2α could be involved in umbilical vasospasm observed in preeclampsia.

CysLT2 receptor-mediated AQP4 up-regulation is involved in ischemic-like injury through activation of ERK and p38 MAPK in rat astrocytes

AimsWe previously reported that cysteinyl leukotriene receptor 2 (CysLT2) mediates ischemic astrocyte injury, and leukotriene D4-activated CysLT2 receptor up-regulates the water channel aquaporin 4 (AQP4). Here we investigated the mechanism underlying CysLT2 receptor-mediated ischemic astrocyte injury induced by 4-h oxygen-glucose deprivation and 24-h recovery (OGD/R). Main methodsPrimary cultures of rat astrocytes were treated by OGD/R to construct the cell injury model. AQP4 expression was inhibited by small interfering RNA (siRNA). The expressions of AQP4 and CysLTs receptors, and the MAPK signaling pathway were determined. Key findingsOGD/R induced astrocyte injury, and increased expression of the CysLT2 (but not CysLT1) receptor and AQP4. OGD/R-induced cell injury and AQP4 up-regulation were inhibited by a CysLT2 receptor antagonist (Bay cysLT2) and a non-selective CysLT receptor antagonist (Bay u9773), but not by a CysLT1 receptor antagonist (montelukast). Knockdown of AQP4 by siRNA attenuated OGD/R injury. Furthermore, OGD/R increased phosphorylation of ERK1/2 and p38, whose inhibitors relieved the cell injury and AQP4 up-regulation. SignificanceThe CysLT2 receptor mediates AQP4 up-regulation in astrocytes, and up-regulated AQP4 leads to OGD/R-induced injury, which results from activation of the ERK1/2 and p38 MAPK pathways.

Pranlukast prevents cysteinyl leukotriene-induced emesis in the least shrew ( Cryptotis parva)

Many chemotherapeutic agents activate multiple signaling systems, including potentially emetogenic arachidonic acid metabolites. Of these messengers, the emetic role of the leukotriene family has been neglected. The aims of this study were to test the emetic potential of key leukotrienes (LTA 4, LTB 4, LTF 4, and the cysteinyl leukotrienes LTC 4, LTD 4 and LTE 4), and to investigate whether the leukotriene CysLT 1 receptor antagonist pranlukast or mixed leukotriene CysLT 1/2 receptor antagonist Bay u9773 can prevent the LTC 4-induced emesis. Least shrews were injected with varying doses of one of the six tested leukotrienes and vomiting parameters were measured for 30 min. LTC 4 and LTD 4 were most efficacious, and significantly increased both the frequency and percentage of animals vomiting at doses from 0.1 and 0.05 mg/kg, respectively. The other tested leukotrienes were either weakly emetic or ineffective at doses up to 4 mg/kg. The relative emetogenic activities of the cysteinyl leukotrienes (LTC 4 = LTD 4 > LTE 4) suggest that leukotriene CysLT 2 receptors have a key role in emesis. However, pranlukast dose-dependently, and at 10 mg/kg completely, blocked LTC 4-induced vomiting, implicating a leukotriene CysLT 1 receptor-mediated emetic effect. Bay u9773 dose-dependently reduced the percentage of animals vomiting, but did not significantly reduce vomiting frequency. Fos immunoreactivity, measured subsequent to LTC 4-induced vomiting to define its putative anatomical substrates, was significantly increased in the enteric nervous system and medullary dorsal vagal complex following LTC 4 ( P < 0.05) versus vehicle injections. This study is the first to show that some leukotrienes induce emesis, possibly involving both central and peripheral leukotriene CysLT 1 and/or leukotriene CysLT 2 receptors.

Method for screening cysteinyl leukotriene receptor 2 antagonists and preliminary screening of compounds

To establish a method for screening cysteinyl leukotriene receptor 2 (CysLT(2)) antagonists and to preliminarily screen a series of synthetic compounds. Rat glioma cell line (C6 cells) highly expressing CysLT(2) receptor was used. Intracellular calcium concentration was measured after stimulation with the agonist LTD(4),which was used to screen compounds with antagonist activity for CysLT(2) receptor. Bay u9773, a CysLT1/CysLT(2) receptor non-selective antagonist, and AP-100984, a CysLT(2) receptor antagonist, were used as control. PT-PCR showed a higher expression of CysLT(2) receptor in C6 cells. LTD(4) at 1 mumol/L significantly increased intracellular calcium in C6 cells; the maximal effect was about 37.5% of ATP, a positive stimulus.LTD(4)-induced increase of intracellular calcium was blocked by CysLT(2) receptor antagonists, but not by CysLT(1) receptor antagonists. Among the synthetic compounds, D(XW-)1,2,13,23,29 and 30 inhibited LTD(4)-induced increase of intracellular calcium. LTD(4)-induced change in intracellular calcium in C6 cells can be used as a screening method for CysLT(2) receptor antagonists. The compounds, D(XW-)1,2,13,23,29 and 30, possess antagonist activity for CysLT(2) receptor.

Leukotriene Pathways and In Vitro Adenotonsillar Cell Proliferation in Children With Obstructive Sleep Apnea

The abundant expression of leukotrienes (LTs) and their receptors in adenotonsillar tissues of children with obstructive sleep apnea (OSA) suggest that LT antagonists could be useful in treating OSA. The effects of LTD4 and of LT receptor antagonists zileuton, montelukast, and BAY u9773 were examined on mixed cell cultures prepared from dissociated tonsils or adenoids harvested intraoperatively from children with polysomnographically diagnosed OSA. Proliferation was assessed by (3)[H]-thymidine incorporation, and inflammatory cytokine production (tumor necrosis factor [TNF]-alpha, interleukin [IL]-6, IL-8, IL-10, and IL-12) was assessed in supernatants using enzyme-linked immunosorbent assay. LTD4 elicited dose-dependent increases in adenotonsillar cell proliferation (p < 0.001; n = 12). All LT antagonists exhibited dose-dependent reductions in adenotonsillar cellular proliferation rates, with montelukast more than BAY u9773 more than zileuton (n = 14/group; p < 0.001). However, BAY u9773 showed partial agonist effects and increased cellular proliferation at higher concentrations (10(-4) mmol/L; p < 0.01; n = 12). LTD4 effects were partially blocked by montelukast and BAY u9773 but not by zileuton. All three antagonists reduced TNF-alpha, IL-6, and IL-12 concentrations, with selective changes in IL-8 and no effects on IL-10 levels. LT pathways mediate intrinsic proliferative and inflammatory signaling pathways in adenotonsillar tissues from children with OSA, and targeted pharmacologic disruption of these pathways may provide nonsurgical alternatives for prevention and treatment of this disease.

Molecular Basis for Reduced Estrone Sulfate Transport and Altered Modulator Sensitivity of Transmembrane Helix (TM) 6 and TM17 Mutants of Multidrug Resistance Protein 1 (ABCC1)

Multidrug resistance protein 1 (MRP1) confers drug resistance and also mediates cellular efflux of many organic anions. MRP1 also transports glutathione (GSH); furthermore, this tripeptide stimulates transport of several substrates, including estrone 3-sulfate. We have previously shown that mutations of Lys(332) in transmembrane helix (TM) 6 and Trp(1246) in TM17 cause different substrate-selective losses in MRP1 transport activity. Here we have extended our characterization of mutants K332L and W1246C to further define the different roles these two residues play in determining the substrate and inhibitor specificity of MRP1. Thus, we have shown that TM17-Trp(1246) is crucial for conferring drug resistance and for binding and transport of methotrexate, estradiol glucuronide, and estrone 3-sulfate, as well as for binding of the tricyclic isoxazole inhibitor N-[3-(9-chloro-3-methyl-4-oxo-4H-isoxazolo-[4,3-c]quinolin-5-yl)-cyclohexylmethyl]-benzamide (LY465803). In contrast, TM6-Lys(332) is important for enabling GSH and GSH-containing compounds to serve as substrates (e.g., leukotriene C(4)) or modulators (e.g., S-decyl-GSH, GSH disulfide) of MRP1 and, further, for enabling GSH (or S-methyl-GSH) to enhance the transport of estrone 3-sulfate and increase the inhibitory potency of LY465803. On the other hand, both mutants are as sensitive as wild-type MRP1 to the non-GSH-containing inhibitors (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK571), 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazol-5-yl)butoxy]phenyl]-ethanone (LY171883), and highly potent 6-[4'-carboxyphenylthio]-5[S]-hydroxy-7[E], 11[Z]14[Z]-eicosatetrenoic acid (BAY u9773). Finally, the differing abilities of the cysteinyl leukotriene derivatives leukotriene C(4), D(4), and F(4) to inhibit estradiol glucuronide transport by wild-type and K332L mutant MRP1 provide further evidence that TM6-Lys(332) is involved in the recognition of the gamma-Glu portion of substrates and modulators containing GSH or GSH-like moieties.

Cysteinyl Leukotriene Receptor Type 1 (CysLT 1) Mediates Contraction of the Guinea Pig Lower Esophageal Sphincter

Objective Leukotriene D 4 (LTD 4) causes contraction of the cat lower esophageal sphincter. The effects of leukotrienes in the guinea pig lower esophageal sphincter and the cysteinyl leukotriene receptor (CysLT) subtype that mediate this contraction are not known. The purpose of the present study was to characterize the CysLT receptors in the guinea pig lower esophageal sphincter. Materials and Methods We measured the contractions of transverse strips from the guinea pig lower esophageal sphincter caused by cysteinyl leukotrienes, LTC 4, LTD 4 and LTE 4, and the dihydroxy leukotriene, LTB 4. We also measured LTD 4-induced contraction inhibited by CysLT receptor antagonists, tetrodotoxin and atropine. Results In the guinea pig lower esophageal sphincter strips, LTC 4, LTD 4 and LTE 4, but not LTB 4, caused concentration-dependent contractions. The relative potencies for cysteinyl leukotrienes to cause contraction were LTD 4 = LTC 4 > LTE 4. LTE 4 was a partial agonist. The LTD 4-induced contraction was inhibited by two selective CysLT 1 receptor antagonists, montelukast and zafirlukast, and by the dual CysLT 1 and CysLT 2 receptor antagonist BAY u9773. The combination of both montelukast and BAY u9773 did not potentiate the inhibition caused by montelukast alone. These findings indicate that CysLT 1 mediates the contraction in the lower esophageal sphincter. Furthermore, LTD 4-induced contraction was not affected by tetrodotoxin or atropine, suggesting a direct effect. Conclusion These results demonstrate that cysteinyl leukotrienes, but not the dihydroxy leukotriene LTB 4, cause contraction of the guinea pig lower esophageal sphincter. The CysLT 1 receptor mediates this contraction.

Leukotriene D4 Stimulates the Migration but Not Proliferation of Endothelial Cells Mediated by the Cysteinyl Leukotriene CysLT1 Receptor via the Extracellular Signal-Regulated Kinase Pathway

The actions of cysteinyl leukotrienes (CysLTs) are mediated by activating CysLT receptors, CysLT1, and CysLT2. The CysLT1 receptor mediates vascular responses to CysLTs; however, its effect on the proliferation and migration of endothelial cells is not clarified. To determine this effect, we observed proliferation and migration in EA.hy926 cells, a human endothelial cell line, and the involvement of activation of mitogen-activated protein kinases (MAPKs). We found that LTD4 did not affect the proliferation, but significantly stimulated the migration of endothelial cells. LTD4 also induced the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, but not those of p38 or JNK. The LTD4-induced migration and ERK1/2 phosphorylation were blocked by the CysLT1-receptor antagonist montelukast and the dual antagonist Bay u9773, but not by the CysLT2-receptor antagonist Bay cysLT2; the migration was also inhibited by the ERK1/2 inhibitor U0126. Our findings indicate that LTD4 stimulates the CysLT1 receptor–mediated migration of endothelial cells; this may be regulated by the ERK1/2 pathway.

Cysteinyl leucotriene receptor type 1 mediates contraction in human and guinea‐pig oesophagus

Leucotriene D(4) (LTD(4)) causes contraction of the guinea-pig and cat oesophagus. Effects of cysteinyl leucotrienes in the human oesophagus were unknown. To investigate and compare the cysteinyl leucotriene effects in the human oesophagus with those in the guinea-pig oesophagus, we measured contraction of muscularis mucosae strips isolated from the human and guinea-pig oesophagus caused by cysteinyl leucotrienes, LTC(4), LTD(4) and LTE(4), as well as the dihydroxy leucotriene, LTB(4). Effects of leucotrienes in human were similar to those in guinea-pig oesophagus. LTC(4) and LTD(4) caused moderate, whereas LTE(4) caused mild, concentration-dependent contraction. LTE(4) was a partial agonist. In contrast, LTB(4) did not cause any contraction. The relative potencies for cysteinyl leucotrienes to cause contraction were LTD(4) = LTC(4) > LTE(4). The LTD(4)-induced contraction was moderately inhibited by two selective CysLT(1) receptor antagonists, montelukast and zafirlukast, in both human and guinea-pig oesophagus. In addition, the LTD(4)-induced contraction was not and only slightly inhibited by BAY u9773, the CysLT(1) and CysLT(2) receptor antagonist, in the human and guinea-pig oesophageal muscularis mucosae respectively. These indicate the existence of the CysLT(1) mediating oesophageal contraction in both human and guinea-pig oesophagus. The LTD(4)-induced contraction was not affected by tetrodotoxin, atropine or capsaicin, suggesting a direct effect. These results demonstrate that cysteinyl leucotrienes but not the dihydroxy leucotriene cause contraction in the human and guinea-pig oesophagus. CysLT(1) mediates contraction in both human and guinea-pig oesophagus.

Synthesis of tenascin and laminin beta2 chain in human bronchial epithelial cells is enhanced by cysteinyl leukotrienes via CysLT1 receptor

BackgroundCysteinyl leukotrienes (CysLTs) are key mediators of asthma, but their role in the genesis of airway remodeling is insufficiently understood. Recent evidence suggests that increased expression of tenascin (Tn) and laminin (Ln) β2 chain is indicative of the remodeling activity in asthma, but represents also an example of deposition of extracellular matrix, which affects the airway wall compliance. We tested the hypothesis that CysLTs affect production of Tn and Ln β2 chain by human bronchial epithelial cells and elucidated, which of the CysLT receptors, CysLT1 or CysLT2, mediate this effect.MethodsCultured BEAS-2B human bronchial epithelial cells were stimulated with leukotriene D4 (LTD4) and E4 (LTE4) and evaluated by immunocytochemistry, Western blotting, flow cytometry, and RT-PCR. CysLT receptors were differentially blocked with use of montelukast or BAY u9773.ResultsLTD4 and LTE4 significantly augmented the expression of Tn, whereas LTD4, distinctly from LTE4, was able to increase also the Ln β2 chain. Although the expression of CysLT2 prevailed over that of CysLT1, the up-regulation of Tn and Ln β2 chain by CysLTs was completely blocked by the CysLT1-selective antagonist montelukast with no difference between montelukast and the dual antagonist BAY u9773 for the inhibitory capacity.ConclusionThese findings suggest that the CysLT-induced up-regulation of Tn and Ln β2 chain, an important epithelium-linked aspect of airway remodeling, is mediated predominantly by the CysLT1 receptor. The results provide a novel aspect to support the use of CysLT1 receptor antagonists in the anti-remodeling treatment of asthma.

Activation of CysLT receptors induces astrocyte proliferation and death after oxygen–glucose deprivation

We recently found that 5-lipoxygenase (5-LOX) is activated to produce cysteinyl leukotrienes (CysLTs), and CysLTs may cause neuronal injury and astrocytosis through activation of CysLT(1) and CysLT(2) receptors in the brain after focal cerebral ischemia. However, the property of astrocyte responses to in vitro ischemic injury is not clear; whether 5-LOX, CysLTs, and their receptors are also involved in the responses of ischemic astrocytes remains unknown. In the present study, we performed oxygen-glucose deprivation (OGD) followed by recovery to induce ischemic-like injury in the cultured rat astrocytes. We found that 1-h OGD did not injure astrocytes (sub-lethal OGD) but induced astrocyte proliferation 48 and 72 h after recovery; whereas 4-h OGD moderately injured the cells (moderate OGD) and led to death 24-72 h after recovery. Inhibition of phospholipase A(2) and 5-LOX attenuated both the proliferation and death. Sub-lethal and moderate OGD enhanced the production of CysLTs that was inhibited by 5-LOX inhibitors. Sub-lethal OGD increased the expressions of CysLT(1) receptor mRNA and protein, while moderate OGD induced the expression of CysLT(2) receptor mRNA. Exogenously applied leukotriene D(4) (LTD(4)) induced astrocyte proliferation at 1-10 nM and astrocyte death at 100-1,000 nM. The CysLT(1) receptor antagonist montelukast attenuated astrocyte proliferation, the CysLT(2) receptor antagonist BAY cysLT2 reversed astrocyte death, and the dual CysLT receptor antagonist BAY u9773 exhibited both effects. In addition, LTD(4) (100 nM) increased the expression of CysLT(2) receptor mRNA. Thus, in vitro ischemia activates astrocyte 5-LOX to produce CysLTs, and CysLTs result in CysLT(1) receptor-mediated proliferation and CysLT(2) receptor-mediated death.

Effects of cysteinyl receptor agonist and antagonists on rat primary cortical neurons

To determine the effect of cysteinyl receptor agonist leukotriene D(4) (LTD(4)) and its antagonists on rat primary neurons. In the primarily cultured rat cortical neurons, the neuron injury was evaluated by measuring intracellular calcium, 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) reduction, and propidium iodide (PI) and Hoechst 33258 staining. The in vitro ischemic injury was induced by oxygen-glucose deprivation (OGD) for 1.5 h and reperfusion for 24 h. LTD(4) (0.01-1 micromol/L) did not induce the elevation of intracellular calcium, neuron viability changes and neuron death. OGD-induced injury was not significantly ameliorated by the CysLT(1) receptor antagonists, pranlukast (0.2-10 micromol/L) and montelukast (0.2-10 micromol/L), as well as by the CysLT(1)/CysLT(2) receptor non-selective antagonist, BAY u9773 (0.02-1 micromol/L). Neither agonist nor antagonists of cysteinyl receptors have the effects on the viability and ischemic-like injury in rat primary neurons.

Molecular mechanisms of the synergy between cysteinyl-leukotrienes and receptor tyrosine kinase growth factors on human bronchial fibroblast proliferation

We have reported that cysteinyl-leukotrienes (cys-LTs) synergise not only with epidermal growth factor (EGF) but also with platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) to induce mitogenesis in human bronchial fibroblasts. We now describe the molecular mechanisms underlying this synergism. Mitogenesis was assessed by incorporation of [3H]thymidine into DNA and changes in protein phosphorylation by Western blotting. Surprisingly, no CysLT receptor antagonists (MK-571, montelukast, BAY u9773) prevented the synergistic mitogenesis. LTD4 did not cause phosphorylation of EGFR nor did it augment EGF-induced phosphorylation of EGFR, and the synergy between LTD4 and EGF was not blocked by the metalloproteinase inhibitor GM6001 or by an HB-EGF neutralising antibody. The EGFR-selective kinase inhibitor, AG1478, suppressed the synergy by LTD4 and EGF, but had no effect on the synergy with PDGF and FGF. While inhibitors of mitogen-activated protein kinase, phosphatidylinositol 3-kinase and protein kinase C (PKC) prevented the synergy, these drugs also inhibited mitogenesis elicited by EGF alone. In contrast, pertussis toxin (PTX) efficiently inhibited the potentiating effect of LTD4 on EGF-induced mitogenesis, as well as that provoked by PDGF or FGF, but had no effect on mitogenesis elicited by the growth factors alone. Whereas LTD4 alone did not augment phosphorylation of extracellular signal-regulated kinase (Erk)-1/2 and Akt, it increased phosphorylation of PKC in a Gαi-dependent manner. Addition of LTD4 prolonged the duration of EGF-induced phosphorylation of Erk-1/2 and Akt, both of which were sensitive to PTX. The effect of cys-LTs involves a PTX-sensitive and PKC-mediated intracellular pathway leading to sustained growth factor-dependent phosphorylation of Erk-1/2 and Akt.

Cysteinyl leukotrienes synergize with growth factors to induce proliferation of human bronchial fibroblasts

Cysteinyl leukotrienes (cys-LTs) are potent asthma-related mediators that function through their G protein-coupled receptors, cys-LT receptor type 1 (CysLT1R) and cys-LT receptor type 2 (CysLT2R). Because many G protein-coupled receptors transactivate the epidermal growth factor receptor (EGFR) through metalloprotease-mediated ligand shedding, we investigated the effects of cys-LTs on signal transduction and proliferation of bronchial fibroblasts. Human bronchial fibroblasts were grown from biopsy specimens of healthy subjects. Mitogenesis was assessed on the basis of tritiated methylthymidine incorporation. Leukotriene (LT) D(4) alone did not increase mitogenesis but dose-dependently increased thymidine incorporation and cell proliferation in the presence of epidermal growth factor (EGF). The enhancement was not prevented by CysLT1R antagonists (MK-571 and montelukast) or by a dual antagonist (BAY u9773), which is consistent with the lack of detectable mRNA for CysLT1R and CysLT2R in bronchial fibroblasts. LTD(4) did not cause EGFR transphosphorylation nor was the synergism blocked by the metalloprotease inhibitor GM6001. The EGFR-selective kinase inhibitor AG1478 suppressed the synergy between LTD(4) and EGF but had no effect on synergistic interactions of LTD(4) with other receptor tyrosine kinase growth factors. The effect of LTD(4) involved a pertussis toxin-sensitive and protein kinase C-mediated intracellular pathway, leading to sustained growth factor-dependent phosphorylation of extracellular signal-regulated kinase 1/2 and protein kinase B (PKB/Akt). Cys-LTs do not transactivate EGFR but have a broader capability to synergize with receptor tyrosine kinase pathways. This study implies a critical role of cys-LTs in airway fibrosis in asthma and other chronic airway diseases, which might not be blocked by therapy with current LT receptor antagonists.

Leukotriene D 4 induces brain edema and enhances CysLT 2 receptor-mediated aquaporin 4 expression

Cysteinyl leukotrienes (including LTC 4, LTD 4, and LTE 4), potent inflammatory mediators, can induce brain-blood barrier (BBB) disruption and brain edema. These reactions are mediated by their receptors, CysLT 1 and CysLT 2 receptors. On the other hand, aquaporin 4 (AQP4) primarily modulates brain water homeostasis and edema after various injuries. Here, we aimed to determine whether AQP4 is involved in LTD 4-induced brain edema. LTD 4 (1 ng in 0.5 μl PBS) microinjection into the cortex increased endogenous IgG exudation (BBB disruption) and water content (brain edema), and enhanced AQP4 expression in mouse brain. The selective CysLT 1 receptor antagonist pranlukast inhibited the IgG exudation, but not the increased water content and AQP4 expression induced by LTD 4. In the cultured rat astrocytes, LTD 4 (10 −9–10 −7 M, for 24 h) similarly enhanced AQP4 expression. The enhanced AQP4 expression was inhibited by Bay u9773, a non-selective CysLT 1/CysLT 2 receptor antagonist, but not by pranlukast. LTD 4 (10 −9–10 −7 M) also induced the mRNA expression of CysLT 2 (not CysLT 1) receptor in astrocytes. These results indicate that LTD 4 modulates brain edema; CysLT 1 receptor mediates vasogenic edema while CysLT 2 receptor may mediate cytotoxic edema via up-regulating AQP4 expression.

Distinct roles of CysLT 1 and CysLT 2 receptors in oxygen glucose deprivation-induced PC12 cell death

Cysteinyl leukotrienes are involved in ischemic brain injury, and their receptors (CysLT 1 and CysLT 2) have been cloned. To clarify which subtype mediates the ischemic neuronal injury, we performed permanent transfection to increase CysLT 1 and CysLT 2 receptor expressions in PC12 cells. Oxygen glucose deprivation (OGD)-induced cell death was detected by Hoechst 33258 and propidium iodide fluorescent staining as well as by flow cytometry. OGD induced late phase apoptosis mainly and necrosis minimally. Over-expression of CysLT 1 receptor decreased and over-expression of CysLT 2 receptor increased OGD-induced cell death. An agonist LTD 4 (10 −7 M) also induced apoptosis, especially in CysLT 2 receptor over-expressing cells. A selective CysLT 1 receptor antagonist montelukast did not affect OGD-induced apoptosis; while non-selective CysLT receptor antagonist Bay u9773 inhibited OGD-induced apoptosis, especially in CysLT 2 receptor over-expressing cells. Thus, CysLT 1 and CysLT 2 receptors play distinct roles in OGD-induced PC12 cell death; CysLT 1 attenuates while CysLT 2 facilitates the cell death.

Vasoconstriction induced by activation of EP 1 and EP 3 receptors in human lung: effects of ONO-AE-248, ONO-DI-004, ONO-8711 or ONO-8713

This study investigated the effects and selectivity of ONO-AE-248, ONO-DI-004, ONO-8711 and ONO-8713 on EP 1 and EP 3 receptors in human pulmonary vessels. The prostanoid receptors involved in the vasoconstriction of human pulmonary arteries (HPA) are TP and EP 3 whereas in pulmonary veins (HPV), this response is associated with TP and EP 1. The experiments were performed in presence of BAY u3405 (TP antagonist). ONO-DI-004 (EP 1 agonist) and ONO-AE-248 (EP 3 agonist), exhibited little or no activity in HPV whereas contractions were induced in HPA with ONO-AE-248. In HPV, the contractions produced with sulprostone (EP 1,3 agonist) were blocked in a non competitive manner by both EP 1 antagonists (ONO-8711, 30 μM; ONO-8713, 10 μM). The involvement of EP 1 mediated contraction in HPV was also observed during the vasorelaxations induced with PGE 1 and 5- cis-carba-PGI 2. In pre-contracted HPV treated with AH6809 (30 μM; EP 1 antagonist) the PGE 1 vasorelaxations were potentiated, while unchanged in HPA. These results demonstrate the selectivity of ONO-AE-248 for the EP 3 receptor in HPA, ONO-DI-004 was ineffective on the EP 1 receptor present in HPV while ONO-8713 was the more potent EP 1 antagonist used in this tissue.