Cysteinyl Leukotriene Receptor 2 is Involved in Inflammation and Neuronal Damage by Mediating Microglia M1/M2 Polarization through NF-κB Pathway.

Microglia activation plays a key role in regulating inflammatory and immune reaction during cerebral ischemia and it exerts pro-inflammatory or anti-inflammatory effect depending on M1/M2 polarization phenotype. Cysteinyl leukotriene 2 receptor (CysLT2R) is a potent inflammatory mediator receptor, and involved in cerebral ischemic injury, but the mechanism of CysLT2R regulating inflammation and neuron damage remains unclear. Here, we found that LPS and CysLT2R agonist NMLTC4 significantly increased microglia proliferation and phagocytosis, up-regulated the mRNA expression of M1 polarization markers (IL-1ß, TNF-α, IFN-γ, CD86 and iNOS), down-regulated the expression of M2 polarization markers (Arg-1, CD206, TGF-ß, IL-10, Ym-1) and increased the release of IL-1ß and TNF-α. CysLT2R selective antagonist HAMI3379 could antagonize these effects. IL-4 significantly up-regulated the mRNA expression of M2 polarization markers, and HAMI3379 further increased IL-4-induced up-regulation of M2 polarization markers expression. Additionally, LPS and NMLTC4 stimulated NF-κB p50 and p65 proteins expression, and promoted p50 transfer to the nucleus. Pre-treatment with HAMI3379 and NF-κB signaling inhibitor Bay 11-7082 could reverse the up-regulation of p50 and p65 proteins expression, and inhibited p50 transfer to the nucleus. The conditional medium of BV-2 cells contained HAMI3379 could inhibit SH-SY5Y cells apoptosis induced by LPS and NMLTC4. These results were further confirmed in primary microglia. The findings indicate that CysLT2R was involved in inflammation and neuronal damage by inducing the activation of microglia M1 polarization and NF-κB pathway, inhibiting microglia M1 polarization and promoting microglia polarization toward M2 phenotype which may exerts neuroprotective effects, and targeting CysLT2R may be a new therapeutic strategy against cerebral ischemia stroke.

Differential signaling of cysteinyl leukotrienes and a novel cysteinyl leukotriene receptor 2 (CysLT2) agonist, N-methyl-leukotriene C4, in calcium reporter and ß arrestin assays.

The cysteinyl leukotrienes (cysLTs) LTC4, LTD4, and LTE4 are lipid mediators with physiological and pathophysiological functions. They exert their effects through G protein-coupled receptors (GPCRs), most notably via CysLT1 and CysLT2 receptor. The roles of the CysLT2 receptor are beginning to emerge. Both LTC4 and LTD4 are potent agonists for the CysLT2 receptor; however, LTC4 is rapidly converted to LTD4, which is also the main endogenous ligand for the CysLT1 receptor. A selective and potent agonist at the CysLT2 receptor would facilitate studies to discern between receptor subtypes. We show here that N-methyl LTC4 (NMLTC4), a metabolically stable LTC4 mimetic, is a potent and selective CysLT2 receptor agonist. Two expression systems were used to evaluate the functional activity of NMLTC4 at human and/or mouse CysLT1 and CysLT2 receptors. Through the aequorin cell-based assay for calcium-coupled GPCRs, NMLTC4 was almost equipotent to LTC4 at CysLT2 receptors but was the least efficacious at CysLT2 receptors. In a ß-galactosidase-ß-arrestin complementation assay, the human (h) CysLT2 receptor can couple with ß-arrestin-2, and NMLTC4 is slightly more potent for eliciting ß-arrestin-2 binding compared with cysLTs. Furthermore, LTE4 is nearly inactive in this assay compared with its weak partial agonist activity in the aequorin system. In a vascular leakage assay, NMLTC4 is potent and active in mice overexpressing hCysLT2 receptor in endothelium, whereas the response is abrogated in CysLT2 receptor knockout mice. Therefore, NMLTC4 is a potent subtype selective agonist for the CysLT2 receptor in vitro and in vivo, and it will be useful to elucidate its biological roles.

A novel glutathione containing eicosanoid (FOG7) chemotactic for human granulocytes.

A biologically active glutathione adduct of the eicosanoid 5-oxo-eicosatetraenoic acid has been observed as a product formed within the murine peritoneal macrophage. This five-oxo glutathione adduct (FOG(7)) was structurally characterized using electrospray tandem mass spectrometry as a 1,4 Michael addition product 5-oxo-7-glutathionyl-8,11,14-eicosatrienoic acid. FOG(7) was found to be highly potent in stimulating eosinophil as well as neutrophil chemotaxis, also capable of initiating actin polymerization, without elevating intracellular free calcium ion concentration within either the eosinophil or polymorphonuclear leukocyte. These biological responses suggest that either FOG(7) activates a subset of receptors mediating the broader biological activity of the parent eicosanoid 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) or that a receptor not activated by 5-oxo-ETE participates in the chemotactic activity of FOG(7). The only other known biologically active glutathione adduct has been leukotriene C(4) (LTC(4)), another eicosanoid that exerts potent effects through the Cys-LT receptor. The biochemical parallel between the formation of LTC(4) and FOG(7) suggests an interesting mechanism by which biologically active eicosanoids derived from electrophilic intermediates may have unique distribution and prolonged efficacy in vivo.

Synthesis of 19-[(2-azido-5-iodo)-benzoyloxy]-LTA4 and enzymatic conversion to the LTC4 analogue.

New photoaffinity probes based on C-19 position of leukotriene A4 has been synthesized from 19-hydroxy-LTA4 methyl ester. Enzymatic conversion into the LTC4 analogue yielded a potential tool for the study of cys-LT2 receptors.

gamma-glutamyl leukotrienase, a gamma-glutamyl transpeptidase gene family member, is expressed primarily in spleen.

We have recently identified a mouse enzyme termed gamma-glutamyl leukotrienase (GGL) that converts leukotriene C4 (LTC4) to leukotriene D4 (LTD4). It also cleaves some other glutathione (GSH) conjugates, but not GSH itself (Carter, B. Z., Wiseman, A. L., Orkiszewski, R., Ballard, K. D., Ou, C.-N., and Lieberman, M. W. (1997) J. Biol. Chem. 272, 12305-12310). We have now cloned a full-length mouse cDNA coding for GGL activity and the corresponding gene. GGL and gamma-glutamyl transpeptidase constitute a small gene family. The two cDNAs share a 57% nucleotide identity and 41% predicted amino acid sequence identity. Their corresponding genes have a similar intron-exon organization and are located 3 kilobases apart. A search of Genbank and reverse transcription-polymerase chain reaction analysis failed to identify additional family members. Mapping of the GGL transcription start site revealed that the GGL promoter is TATA-less but contains an initiator, a control element for transcription initiation. Northern blots for GGL expression were negative. As judged by ribonuclease protection, in situ hybridization, and measurement of enzyme activity, spleen had the highest level of GGL expression. GGL is also expressed in thymic lymphocytes, bronchiolar epithelial cells, pulmonary interstitial cells, renal proximal convoluted tubular cells, and crypt cells of the small intestine as well as in cerebral, cerebellar, and brain stem neurons but not in glial cells. GGL is widely distributed in mice, suggesting an important role for this enzyme.

ATP-dependent transport of bilirubin glucuronides by the multidrug resistance protein MRP1 and its hepatocyte canalicular isoform MRP2.

Bilirubin is secreted from the liver into bile mainly as monoglucuronosyl and bisglucuronosyl conjugates. We demonstrate for the first time that ATP-dependent transport of both bilirubin glucuronides is mediated by the multidrug resistance protein (MRP1) as well as by the distinct canalicular (apical) isoform MRP2, also termed cMRP or cMOAT (canalicular multispecific organic anion transporter). In membrane vesicles from MRP1-transfected HeLa cells mono[3H]glucuronosylbilirubin and bis[3H]glucuronosylbilirubin (each at 0.5 microM) were transported with rates of 5.3 and 3.1 pmol/min per mg of protein respectively. Rat hepatocyte canalicular membrane vesicles, which contain Mrp2 (the rat equivalent of MRP2), transported mono[3H]glucuronosylbilirubin and bis[3H]glucuronosylbilirubin at rates of 8.9 and 8.5 pmol/min per mg of protein, whereas membrane vesicles from mutant liver lacking Mrp2 showed no transport of the conjugates. In membrane vesicles from human hepatoma Hep G2 cells, which predominantly expressed MRP2, transport rates were 8.3 and 4.4 pmol/min per mg of protein for monoglucuronosylbilirubin and bisglucuronosylbilirubin respectively. ATP-dependent transport of the glutathione S-conjugate -3H-leukotriene C4, an established high-affinity substrate for MRP1 and MRP2, was inhibited by both bilirubin glucuronides with IC50 values between 0.10 and 0.75 microM. The ratios of leukotriene C4 transport and bilirubin glucuronide transport, determined in the same membrane vesicle preparation, indicated substrate specificity differences between MRP1 and MRP2 with a preference of MRP2 for the glucuronides.

Glutathione conjugates recognize the Rossmann fold of glyceraldehyde-3-phosphate dehydrogenase.

Leukotriene (LT) C4 and other glutathione conjugates are synthesized intracellularly and then move to the plasma membrane for export. The intracellular proteins that bind these molecules and the significance of these interactions are poorly understood. To identify the binding sites of membrane-associated proteins that recognize these molecules, we utilized photoaffinity probes to label the inner leaflet of erythrocytes. The predominant molecule labeled with S-(p-nitrobenzyl)glutathione-[125I]4-azidosalicylic acid (PNBG-[125I]ASA) or LTC4-[125I]4-azidosalicylic acid (LTC4-[125I]ASA) was 38 kDa. The protein was labeled with PNBG-[125I]ASA, electroblotted to polyvinylidene difluoride membranes, digested in situ with lysyl endopeptidase, and two radiolabeled peptides isolated by reverse phase-high performance liquid chromatography. These contained an identity of 7/11 with amino acids 119-129, and 11/11 with amino acids 67-77 of human liver glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. Photoaffinity labeling with PNBG-[125I]ASA was blocked completely by 100 microM ATP and greater than 50% with 100 microM NAD+. LTC4-[125I]ASA binding to the NAD+ site was confirmed by V8 protease digestion of purified GAPDH labeled with LTC4-[125I]ASA or PNBG-[125I]ASA, with both labels localized to the 6.8-kDa N-terminal fragment. Photoaffinity labeling of HL-60 cells with LTC4-125I-ASA identified GAPDH as the predominant cytoplasmic binding protein in these cells. These data indicate that GAPDH is a membrane-associated and cytoplasmic protein which binds glutathione conjugates including LTC4.

Cardiovascular effects of N-methyl leukotriene C4, a nonmetabolizable leukotriene C4 analogue, and the antagonism of leukotriene-induced hypotension by Ro 23-3544, in the American bullfrog, Rana catesbeiana.

Although some leukotriene antagonists have been reported to block leukotriene (LT) C4 responses in vivo, it is difficult to determine whether those antagonists block the effect of LTC4 directly or act via blocking the action of LTD4, as LTC4 is metabolized to LTD4 rapidly in vivo. In this study, the dose-response curves of N-methyl LTC4 (NMLTC4), the nonmetabolizable LTC4 analogue, and the peptidoleukotrienes (LTC4, LTD4, and LTE4) were obtained in the absence and presence of the leukotriene antagonist Ro 23-3544 in cannulated frogs. The more potent effect of NMLTC4 suggests that receptors that preferentially bind LTC4 exist in frog vascular smooth muscle and the previously reported LTC4 effect is a combination of LTC4 and its less potent metabolite LTD4. The NMLTC4- and LTC4-induced hypotensive effects were antagonized by Ro 23-3544. Ro 23-3544 also antagonized the effects induced by high doses of LTD4 and LTE4. Ro 23-3544 had no effect on duration of response and did not affect heart rate responses to LTC4 at low dose of the antagonist. The data suggest that receptors that preferentially bind LTC4 in bullfrog vascular smooth muscle regulate the hypotensive effect and that they can be antagonized by Ro 23-3544.

Leukotriene C4-stimulated contractions in bullfrog lung are affected by cold acclimation and calcium antagonists.

Leukotriene C4 (LTC4) contracts isolated bullfrog lung. This study examined effects of cold-acclimation and the involvement of extracellular and intracellular Ca++ on the contractile response to LTC4. The response to LTC4 was greater in lungs from warm-acclimated (22 degrees C) frogs compared with cold-acclimated (5 degrees C) frogs at incubation temperatures of both 22 degrees C and 5 degrees C. LTC4, LTC5, and N-methyl LTC4 were equally effective in stimulating lung contraction at concentrations from 1-100 nM. Nicardipine (3 microM) partially antagonized the response to LTC4, but verapamil, nifedipine, or nitrendipine at the same concentration was ineffective. Ethylene glycol tetraacetic acid (EGTA, 0.3 mM) prevented the response to 30 nM LTC4, but the response was restored when the lung was retested in EGTA-free medium containing Ca++, suggesting that extracellular Ca++ was involved in the response. Caffeine (10 mM) or thapsigargin (1 mM) inhibited the responses to LTC4, suggesting a role for intracellular Ca++ in the contraction.

Glutathione S-transferase-dependent conjugation of leukotriene A4-methyl ester to leukotriene C4-methyl ester in mammalian skin.

The glutathione S-transferase (GST)-dependent conjugation of reduced glutathione (GSH) with leukotriene A4 (LTA4)-methyl ester in rodent and human skin was investigated. Incubation of [3H]LTA4-methyl ester (1 nmole, approximately 200,000 dpm) with cytosol prepared from rat, mouse and human skin or with affinity purified GST from rat skin cytosol in the presence of GSH resulted in the formation of LTC4-methyl ester. Maximum enzyme activity was observed in rat skin followed by mouse and human skin. With heat-denatured cytosol or in the absence of GSH, the product formation was negligible. GST purified from rat skin cytosol by GSH-agarose affinity chromatography exhibited a several-fold increase in the specific activity of enzyme with 1-chloro-2,4-dinitrobenzene (55-fold), ethacrynic acid (67-fold) and LTA4-methyl ester (12-fold) as substrates. Western blot analysis of the affinity purified GST indicated a predominant expression of the Pi class of GST isozyme followed by Mu and Alpha classes of isozymes. The formation of LTC4-methyl ester was established by its radioactivity profile on high pressure liquid chromatography and absorption spectroscopy. These results suggest that, in addition to xenobiotic metabolism, cutaneous GSTs may also be capable of metabolizing physiological substrates such as LTA4.

The pharmacology of N-methyl LTC4; a metabolically stable LTC4-mimetic.

N-methyl LTC4 (NMLTC4) a synthetic analogue of LTC4, has been shown not to be a substrate for gamma-glutamyl transpeptidase. NMLTC4 produced contractions of the guinea pig ileum and trachea with pD2 values of 7.7 +/- 0.12 (n = 6) and 8.1 +/- 0.1 (n = 6) respectively, compared with values of 9.0 +/- 0.1 (n = 5) and 8.0 +/- 0.2 (n = 6) for LTC4. The concentration-response curve to LTC4 and NMLTC4 on ileum was displaced to the right by FPL55712. The corresponding pA2 values were 6.3 +/- 0.3 (n = 10) for LTC4 and 5.7 +/- 0.2 (n = 6) for NMLTC4. In the presence of acivicin, a gamma-glutamyl transpeptidase inhibitor, the LTC4 concentration-response curve on trachea was displaced to the left, but the NMLTC4 curve was unaffected. The comparative potencies in the presence of acivicin on trachea indicate that LTC4 is approximately 6 times more potent than NMLTC4 whereas on ileum, in the presence of FPL55712 LTC4 is approximately 14 times more potent. In-vivo NMLTC4 is a weak bronchoconstrictor substance being 20-30 less potent than LTC4. However, unlike the in-vitro studies the bronchospasm was significantly reduced by pretreatment with LTD4 antagonists. NMLTC4 administered intravenously produced a pronounced hypertensive effect which appeared to be due to peripheral vasoconstriction.

Racemic LTA4 methyl ester bioconversion into LTC4 methyl ester by various glutathione S-transferases.

It has been shown that various glutathione transferases can synthesize leukotriene C4, or its methyl ester, from glutathione and leukotriene A4. We questioned whether the same enzymes could be used to resolve racemic leukotriene A4 methyl ester (more easily prepared than the optically active enantiomer) and to produce leukotriene C4 methyl ester selectively. We present in this paper a study of the enantioselectivity of some rat liver glutathione transferase isozymes and of the glutathione transferase of human placenta for the leukotriene A4 methyl ester isomers. The rat liver 3-4 glutathione transferase exhibited the highest conversion rate but preferentially converted the (5R, 6R) leukotriene A4 methyl ester. The placental enzyme was fairly selective for the natural (5S, 6S) enantiomer but the rate of conversion was low.

Transformation of leukotriene A4 methyl ester to leukotriene C4 monomethyl ester by cytosolic rat glutathione transferases.

Six major basic cytosolic glutathione transferases from rat liver catalyzed the conversion of leukotriene A4 methyl ester to the corresponding leukotriene C4 monomethyl ester. Glutathione transferase 4-4, the most active among these enzymes, had a Vmax of 615 nmol X min-1 X mg protein-1 at 30 degrees C in the presence of 5 mM glutathione. It was followed in efficiency by transferase 3-4 which had a Vmax of 160 nmol X min-1 X mg-1 under the same conditions. Transferases 1-1, 1-2, 2-2 and 3-3 had at least 30 times lower Vmax values than transferase 4-4.