Activation of calcium- and voltage-gated potassium channels of large conductance by leukotriene B4.

Calcium/voltage-gated, large conductance potassium (BK) channels control numerous physiological processes, including myogenic tone. BK channel regulation by direct interaction between lipid and channel protein sites has received increasing attention. Leukotrienes (LTA4, LTB4, LTC4, LTD4, and LTE4) are inflammatory lipid mediators. We performed patch clamp studies in Xenopus oocytes that co-expressed BK channel-forming (cbv1) and accessory ß1 subunits cloned from rat cerebral artery myocytes. Leukotrienes were applied at 0.1 nm-10 µm to either leaflet of cell-free membranes at a wide range of [Ca(2+)]i and voltages. Only LTB4 reversibly increased BK steady-state activity (EC50 = 1 nm; Emax reached at 10 nm), with physiological [Ca(2+)]i and voltages favoring this activation. Homomeric cbv1 or cbv1-ß2 channels were LTB4-resistant. Computational modeling predicted that LTB4 docked onto the cholane steroid-sensing site in the BK ß1 transmembrane domain 2 (TM2). Co-application of LTB4 and cholane steroid did not further increase LTB4-induced activation. LTB4 failed to activate ß1 subunit-containing channels when ß1 carried T169A, A176S, or K179I within the docking site. Co-application of LTB4 with LTA4, LTC4, LTD4, or LTE4 suppressed LTB4-induced activation. Inactive leukotrienes docked onto a portion of the site, probably preventing tight docking of LTB4. In summary, we document the ability of two endogenous lipids from different chemical families to share their site of action on a channel accessory subunit. Thus, cross-talk between leukotrienes and cholane steroids might converge on regulation of smooth muscle contractility via BK ß1. Moreover, the identification of LTB4 as a highly potent ligand for BK channels is critical for the future development of ß1-specific BK channel activators.

Vitamin E, γ-tocopherol, diminishes ex vivo basophil response to dust mite allergen.

Epidemiologic studies suggest that dietary vitamin E is a candidate intervention for atopic disease. We used in vitro and ex vivo exposures to test the hypothesis that the most common dietary isoform of vitamin E, γ-tocopherol (γT), could suppress FcεRI-mediated basophil activation. Rat basophilic leukemia (RBL)-SX38 cells that express human FcεRI were treated with or without γT, followed by stimulation with α-IgE. In the ex vivo study, 20 Der f 1-allergic volunteers consumed a γT-enriched supplement for 7 days. Their basophils were challenged ex vivo with α-IgE and graded doses of Der f 1 before and after the supplementation period. γt treatment of RBL-SX38 cells significantly reduced basophil degranulation and de novo TH2 cytokine production. Daily consumption of a γT-rich supplement by dust mite-allergic volunteers reduced basophil activation after ex vivo dust mite challenge. Vitamin E supplements rich in γT may be useful adjuncts in decreasing atopic disease.

Leukotriene C4 induces migration of human monocyte-derived dendritic cells without loss of immunostimulatory function.

Generation of human monocyte-derived dendritic cells (DCs) for cancer vaccination involves ex vivo maturation in the presence of proinflammatory cytokines and prostaglandin E(2) (PGE(2)). Although the inclusion of PGE(2) during maturation is imperative for the induction of DC migration, PGE(2) has unfavorable effects on the immunostimulatory capacity of these cells. Like PGE(2), leukotrienes (LTs) are potent mediators of DC migration. We therefore sought to characterize the migratory and immunologic properties of DCs that matured in the presence of LTB(4), LTC(4), LTD(4), and PGE(2). Here, we demonstrate that DCs matured in the presence of LTC(4), but not LTB(4) or LTD(4), are superior to PGE(2)-matured DCs in stimulating CD4(+) T-cell responses and in inducing antigen-specific cytotoxic T lymphocytes (CTLs) in vitro without concomitant induction or recruitment of regulatory T cells (Tregs). LTC(4)-matured DCs migrate efficiently through layers of extracellular matrix and secrete higher levels of immunostimulatory IL-12p70 while producing reduced levels of immune-inhibitory IL-10, IL12p40, indoleamine-2,3-dioxidase, and TIMP-1 (tissue inhibitor of matrix metalloproteinases). Intracellular calcium mobilization and receptor antagonist studies reveal that, in contrast to LTD(4), LTC(4) did not signal through CysLTR(1) in DCs. Collectively, our data suggest that LTC(4) represents a promising candidate to replace PGE(2) in DC maturation protocols for cancer vaccination.

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

OBJECTIVE: To establish a method for screening cysteinyl leukotriene receptor 2 (CysLT(2)) antagonists and to preliminarily screen a series of synthetic compounds. METHODS: 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. RESULT: 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. CONCLUSION: 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.

Effect of TA-270, a novel quinolinone derivative, on antigen-induced nasal blockage in a guinea pig model of allergic rhinitis.

TA-270 (4-hydroxy-1-methyl-3-octyloxy-7-sinapinoylamino-2(1H)-quinolinone) is a novel quinolinone derivative that has been demonstrated to possess an anti-oxidative activity against peroxynitrite, a potent oxidant, that is generated by the reaction of nitric oxide with superoxide anions. The current study describes the inhibitory effect of TA-270 on the biphasic nasal blockage induced by repeated antigen challenge in an allergic rhinitis guinea pig model. In the present in vitro study, TA-270 potently inhibited the oxidative reaction induced by peroxynitrite (IC(50)=79 nM). In addition, TA-270 (0.3-30 mg/kg, p.o.) dose-dependently inhibited peroxynitrite (3 mM, 10 mul/nostril)-induced nasal blockage in guinea pigs. In the antigen-induced allergic rhinitis model, TA-270 (0.3, 3, and 30 mg/kg, p.o.) given 1 h before the antigen challenge suppressed early phase nasal blockage by 36%, 42%, and 63%, respectively. Furthermore, TA-270 (0.3, 3, and 30 mg/kg, p.o.) showed a relatively strong suppression of late phase nasal blockage (39%, 62%, and 72%, respectively). The late phase nasal blockage was significantly inhibited (61%) even when TA-270 (30 mg/kg, p.o.) was administered 18 h before the antigen challenge. In conclusion, TA-270 improved antigen-induced nasal blockage, probably through its peroxynitrite scavenging action, and the effect was sustained for at least 18 h. Thus, TA-270 would be expected to relieve nasal blockage in allergic rhinitis patients.

[Effect of shuanglong capsule on content of leukotrienes compound of lung tissues in asthmatic rats].

OBJECTIVE: To observe the changing contents of leukotriene B4 ( LTB4 ), leukotriene C4 ( LTC4 ), and leukotriene D4 (LTD4 ) of lung tissue in asthmatic rats, and explore the effect of Shuanglong Capsule (SLC) on it. METHODS: SD rats were randomly divided into the nomal group, asthmatic model group, Dexamethasone group and the high, middle and low dose SLC groups. All rats except those in the normal group were sensitized by ovalbumin and challenged with the antigen, and the contents of LTB4, LTC4 and LTD4 in lung tissue of all the groups were measured by reverse phase-high performance liquid chromatography (RP-HPLC) and compared. RESULTS: The levels of LTB4, LTC4, and LTD4 of asthmatic rats were significantly higher than those of rats in the normal group. Dexamethasone and SLC at the dose of 8. 27 g/kg or 4. 13 g/kg could significantly inhibit the production of leukotrienes of lung tissue in asthmatic rats (P <0.05). CONCLUSION: SLC can significantly inhibit the formation of inflammatory medium LTs of lung tissue in asthmatic rats, it may be one of the key mechanisms of SLC in anti-asthma and anti-inflammatory action.

Leukotriene D4 mediates survival and proliferation via separate but parallel pathways in the human intestinal epithelial cell line Int 407.

We demonstrated previously that leukotriene D4 (LTD4) regulates proliferation of intestinal epithelial cells through a CysLT receptor by protein kinase C (PKC)epsilon-dependent stimulation of the mitogen-activated protein kinase ERK1/2. Our current study provides the first evidence that LTD4 can activate 90-kDa ribosomal S6 kinase (p90RSK) and cAMP-responsive element-binding protein (CREB) via pertussis-toxin-sensitive Gi protein pathways. Transfection and inhibitor experiments revealed that activation of p90RSK, but not CREB, is a PKCepsilon/Raf-1/ERK1/2-dependent process. LTD4-mediated CREB activation was not affected by expression of kinase-dead p90RSK but was abolished by transfection with the regulatory domain of PKCalpha (a specific dominant-inhibitor of PKCalpha). Kinase-negative mutants of p90RSK and CREB (K-p90RSK and K-CREB) blocked the LTD4-induced increase in cell number and DNA synthesis (thymidine incorporation). Compatible with these results, flow cytometry showed that LTD4 caused transition from the G0/G1 to the S+G2/M cell cycle phase, indicating increased proliferation. Similar treatment of cells transfected with K-p90RSK resulted in cell cycle arrest in the G0/G1 phase, consistent with a role of p90RSK in LTD4-induced proliferation. On the other hand, expression of K-CREB caused a substantial buildup in the sub-G0/G1 phase, suggesting a role for CREB in mediating LTD4-mediated survival in intestinal epithelial cells. Our results show that LTD4 regulates proliferation and survival via distinct intracellular signaling pathways in intestinal epithelial cells.

Leukotriene D(4) activates MAPK through a Ras-independent but PKCepsilon-dependent pathway in intestinal epithelial cells.

We have recently shown that leukotriene D(4) (LTD(4)) increases cell survival in intestinal epithelial cells. Here we report and explore the complementary finding that LTD(4) also enhances proliferation in these cells. This proliferative response was approximately half of that induced by epidermal growth factor (EGF) and its required activation of protein kinase C (PKC), Ras and the mitogen-activated protein kinase (MAPK) Erk-1/2. EGF also activated Erk-1/2 in these cells; however the EGF-receptor inhibitor PD153035 did not affect the LTD(4)-induced activation of Erk-1/2. In addition, LTD(4) did not induce phosphorylation of the EGF receptor, nor did pertussis toxin (PTX) block EGF-induced activation of Erk-1/2, thus refuting a possible crosstalk between the receptors. Furthermore, LTD(4)-induced, but not EGF-induced, activation of Erk-1/2 was sensitive to PTX, PKC inhibitors and downregulation of PKCepsilon. A definite role for PKCepsilon in LTD(4)-induced stimulation of Erk-1/2 was documented by the inability of LTD(4) to activate Erk-1/2 in cells transfected with either the regulatory domain of PKCepsilon (an isoform specific dominant-negative inhibitor) or a kinase-dead PKCepsilon. Although Ras and Raf-1 were both transiently activated by LTD(4), only Raf-1 activation was abolished by abrogation of the PKC signal. Furthermore, the LTD(4)-induced activation of Erk-1/2 was unaffected by transfection with dominant-negative N17 Ras but blocked by transfection with kinase-dead Raf-1. Consequently, LTD(4) regulates the proliferative response by a distinct Ras-independent, PKCepsilon-dependent activation of Erk-1/2 and a parallel Ras-dependent signaling pathway.

The murine cysteinyl leukotriene 2 (CysLT2) receptor. cDNA and genomic cloning, alternative splicing, and in vitro characterization.

Two classes of cysteinyl leukotriene receptor, CysLT(1) and CysLT(2), have been identified and pharmacologically characterized in human tissues. Although the CysLT(1) receptor mediates the proinflammatory effects of leukotrienes in human asthma, the physiological roles of CysLT(2) receptor are not defined, and a suitable mouse model would be useful in delineating function. We report here the molecular cloning and characterization of the mouse CysLT(2) receptor (mCysLT(2)R) from heart tissue. mCysLT(2)R cDNA encodes a protein of 309 amino acids, truncated at both ends compared with the human ortholog (hCysLT(2)R). The gene resides on the central region of mouse chromosome 14 and is composed of 6 exons with the entire coding region located in the last exon. Two 5'-untranslated region splice variants were identified with the short form lacking exon 3 as the predominant transcript. Although the overall expression of mCysLT(2)R is very low, the highest expression was detected in spleen, thymus, and adrenal gland by ribonuclease protection assay, and discrete sites of expression in heart were observed by in situ hybridization. Intracellular calcium mobilization in response to cysteinyl leukotriene administration was detected in human embryonic kidney 293T cells transfected with recombinant mCysLT(2)R with a rank order of potency leukotriene C(4)(LTC(4) ) = LTD(4)>>LTE(4). [(3)H]LTD(4) binding to membranes expressing mCysLT(2)R could be effectively competed by LTC(4) and LTD(4) and only partially inhibited by LTE(4) and BAYu9773. The identification of mCysLT(2)R will be useful for establishing CysLT(2)R-deficient mice and determining novel leukotriene functions.

Identification in mice of two isoforms of the cysteinyl leukotriene 1 receptor that result from alternative splicing.

Two classes of human G protein-coupled receptors, cysteinyl leukotriene 1 (CysLT(1)) and CysLT(2) receptors, recently have been characterized and cloned. Because the CysLT(1) receptor blockers are effective in treating human bronchial asthma and the mouse is often used to model human diseases, we isolated the mouse CysLT(1) receptor from a mouse lung cDNA library and found two isoforms. A short isoform cDNA containing two exons encodes a polypeptide of 339 aa with 87.3% amino acid identity to the human CysLT(1) receptor. A long isoform has two additional exons and an in-frame upstream start codon resulting in a 13-aa extension at the N terminus. Northern blot analysis revealed that the mouse CysLT(1) receptor mRNA is expressed in lung and skin; and reverse transcription-PCR showed wide expression of the long isoform with the strongest presence in lung and skin. The gene for the mouse CysLT(1) receptor was mapped to band XD. Leukotriene (LT) D(4) induced intracellular calcium mobilization in Chinese hamster ovary cells stably expressing either isoform of the mouse CysLT(1) receptor cDNA. This agonist effect of LTD(4) was fully inhibited by the CysLT(1) receptor antagonist, MK-571. Microsomal membranes from each transformant showed a single class of binding sites for [(3)H]LTD(4); and the binding was blocked by unlabeled LTs, with the rank order of affinities being LTD(4) >> LTE(4) = LTC(4) >> LTB(4). Thus, the dominant mouse isoform with the N-terminal amino acid extension encoded by an additional exon has the same ligand response profile as the spliced form and the human receptor.

Reduced inflammatory response in rats fed fat-rich diets: role of leukotrienes.

The effect of fat-rich diets on the acute inflammatory response was examined. Male Wistar rats aged 21 days were fed, for 6 weeks, with a control diet (4% fat content), or a control diet supplemented with coconut or soybean oils (15% fat content). Carrageenan-induced paw oedema and pleurisy were evaluated. Prostaglandin (PG) E2 and leukotriene (LT) C4/D4 concentrations were determined in the pleural exudate (ELISA). Pleural samples were tested for their effect on cutaneous vascular permeability of control rats and the effect of a LTD4 receptor antagonist (L660-711; 10 mg/kg; i.v.) examined. Relative to controls, rats fed both fat-rich diets presented a significant reduction in protein leakage and oedema formation without affecting the number of migrating leukocytes. Production of LTC4/D4 in pleural exudate was significantly increased from 1.8 +/- 0.2 ng/ml in controls to 2.8 +/- 0.2 and 3.0 +/- 0.3 ng/ml in animals fed coconut and soybean oil enriched diets, respectively, without changes in PGE2 production. The activity of these samples on cutaneous vascular permeability was 50% reduced, returning to control values after treatment of testing animals with a LTD4 receptor antagonist. Rats fed fat-rich diets presented a reduced inflammatory response due, at least in part, to the LTC4/D4 mediated vasoconstrictor effect.

Mechanisms involved in the antiinflammatory effect of propolis extract.

Propolis is a natural product produced by the honey bee. The extract contains amino acids, flavanoids, terpenes and cinnamic acid derivatives. In various in vitro models propolis extract was shown to inhibit platelet aggregation and to inhibit eicosanoid synthesis, suggesting that it might have potent antiinflammatory properties. A 13% aqueous extract was tested orally in three dose levels (1, 5 and 10 ml/kg) on the carrageenan rat paw oedema model and on adjuvant-induced arthritis in rats. In both models, the extract showed potent dose-related antiinflammatory activity, which compared well with that of diclofenac (as a reference standard). The extract was then tested on an isolated sensitized guinea pig lung preparation to study its effect on the release of prostaglandins, leukotrienes and histamine. It is concluded that propolis extract has potent antiinflammatory properties in vivo. Its activity can be well correlated with its effects on the release of various mediators of inflammation.