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1.
Biochim Biophys Acta Mol Basis Dis ; 1865(9): 2379-2392, 2019 09 01.
Article in English | MEDLINE | ID: mdl-31167124

ABSTRACT

BACKGROUND: Abnormalities of the L-arginine-nitric oxide pathway induce hypertension. 5-Lipoxygenase (5-LO) is the key enzyme involved in synthesis of leukotrienes (LTs). However, whether nitricoxide synthase dysfunction induces hypertensive vascular remodeling by regulating 5-LO activity and its downstream inflammatory metabolites remains unknown. METHODS AND RESULTS: Six-week L-NAME treatment significantly induced hypertension and vascular remodeling in both wild-type (WT) and 5-LO-knockout (5-LO-KO) mice, and blood pressure in caudal and carotid arteries was lower in 5-LO-KO than WT mice with L-NAME exposure. On histology, L-NAME induced less media thickness, media-to-lumen ratio, and collagen deposition and fewer Ki-67-positive vascular smooth muscle cells (VSMCs) but more elastin expression in thoracic and mesenteric aortas of 5-LO-KO than L-NAME-treated WT mice. L-NAME significantly increased LT content, including LTB4 and cysteinyl LT (CysLTs), in plasma and neutrophil culture supernatants from WT mice. On immunohistochemistry, L-NAME promoted the colocalization of 5-LO and 5-LO-activating protein on the nuclear envelope of cultured neutrophils, which was accompanied by elevated LT content in culture supernatants. In addition, LTs significantly promoted BrdU incorporation, migration and phenotypic modulation in VSMCs. CONCLUSION: L-NAME may activate the 5-LO/LT pathway in immune cells, such as neutrophils, and promote the products of 5-LO metabolites, including LTB4 and CysLTs, which aggravate vascular remodeling in hypertension. 5-LO deficiency may protect against hypertension and vascular remodeling by reducing levels of 5-LO downstream inflammatory metabolites.


Subject(s)
Arachidonate 5-Lipoxygenase/genetics , Hypertension/prevention & control , Vascular Remodeling , Animals , Aorta/metabolism , Aorta/pathology , Arachidonate 5-Lipoxygenase/deficiency , Blood Pressure/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Hypertension/chemically induced , Hypertension/pathology , Leukotriene A4/blood , Leukotriene A4/pharmacology , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , NG-Nitroarginine Methyl Ester/metabolism , NG-Nitroarginine Methyl Ester/toxicity , Neutrophils/immunology , Neutrophils/metabolism , Nitric Oxide Synthase Type II/metabolism , Rats , Rats, Sprague-Dawley , Vascular Remodeling/drug effects
2.
Blood ; 111(4): 1789-96, 2008 Feb 15.
Article in English | MEDLINE | ID: mdl-18042794

ABSTRACT

We previously identified a negative correlation between histamine release to histamine releasing factor/translationally controlled tumor protein (HRF/TCTP) and protein levels of the Src homology 2 domain-containing inositol 5' phosphatase (SHIP) in basophils. We have also demonstrated that HRF/TCTP primes basophils to release mediators. The purpose of this study was to begin characterization of signal transduction events directly induced by HRF/TCTP and to investigate these events when HRF/TCTP is used as a priming agent for human basophil histamine release. Highly purified human basophils were examined for surface expression of bound HRF/TCTP, changes in calcium, and phosphorylation of Akt, mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK), Syk, and FcepsilonRIgamma. Results showed that basophils from all donors bound HRF/TCTP. There was a biphasic calcium response to HRF/TCTP, which corresponded to the magnitude of histamine release. Furthermore, those donors who have direct histamine release when exposed to HRF/TCTP (HRF/TCTP responder [HRF/TCTP-R] donors) have phosphorylation of Syk, Akt, MEK, and ERK. Remarkably, basophils from HRF/TCTP-nonresponder (HRF/TCTP-NR) donors do not show phosphorylation of these molecules. This finding is different from IL-3, which also primes basophils for histamine release, but does show phosphorylation of these events. We conclude that priming induced by HRF/TCTP is distinct from that induced by IL-3.


Subject(s)
Basophils/physiology , Biomarkers, Tumor/physiology , Basophils/cytology , Biomarkers, Tumor/genetics , Calcium/blood , Histamine Release/physiology , Humans , Interleukin-3/physiology , Intracellular Signaling Peptides and Proteins/metabolism , Leukotriene A4/blood , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Receptors, IgE/blood , Recombinant Proteins/metabolism , Signal Transduction , Syk Kinase , Tumor Protein, Translationally-Controlled 1
3.
Clin Ther ; 25(3): 972-9, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12852711

ABSTRACT

BACKGROUND: Leukotriene inhibitors and leukotriene-receptor antagonists are effective in the treatment of inflammatory diseases such as asthma. A search of the entirety of MEDLINE using the terms diet plus leukotrienes identified numerous studies that have explored dietary-management strategies to reduce leukotriene levels through supplementation with polyunsaturated fatty acids such as gamma-linolenic acid (GLA) and eicosapentaenoic acid (EPA). However, the search found no studies on the use of combinations of these fatty acids in patients with asthma. OBJECTIVE: The goal of this study was to determine the effect of daily intake of an emulsion (PLT 3514) containing dietary GLA and EPA on ex vivo stimulated whole blood leukotriene biosynthesis in patients with atopic asthma. METHODS: This was a randomized, double-blind, placebo-controlled, parallel-group, prospective trial in patients with mild to moderate atopic asthma. Patients consumed 10 g PLT 3514 emulsion (containing 0.75 g GLA + 0.5 g EPA), 15 g PLT 3514 emulsion (containing 1.13 g GLA + 0.75 g EPA), or placebo (olive oil) emulsion daily for 4 weeks. Plasma fatty acids were measured by gas chromatography, and stimulated whole blood leukotrienes were measured by reverse-phase high-performance liquid chromatography with ultraviolet detection using a diode array detector. RESULTS: Forty-three patients (33 women, 10 men) participated in the study. Leukotriene biosynthesis was significantly decreased in patients consuming 10 or 15 g PLT 3514 compared with placebo (P < 0.05, analysis of covariance). No clinically significant changes in vital signs were observed throughout the study, and there were no significant between-group differences in treatment-emergent adverse events or mean clinical laboratory values. CONCLUSION: Daily consumption of dietary GLA and EPA in a novel emulsion formulation inhibited leukotriene biosynthesis in this population of patients with atopic asthma and was well tolerated.


Subject(s)
Asthma/therapy , Fatty Acids, Unsaturated/therapeutic use , Food, Formulated , Leukotriene A4/biosynthesis , Adult , Asthma/blood , Double-Blind Method , Eicosapentaenoic Acid/therapeutic use , Emulsions , Fatty Acids/blood , Female , Humans , Leukotriene A4/antagonists & inhibitors , Leukotriene A4/blood , Male , Prospective Studies , gamma-Linolenic Acid/therapeutic use
4.
J Med Chem ; 45(16): 3482-90, 2002 Aug 01.
Article in English | MEDLINE | ID: mdl-12139459

ABSTRACT

Leukotriene B(4) (LTB(4)) is a potent, proinflammatory mediator involved in the pathogenesis of a number of diseases including inflammatory bowel disease, psoriasis, rheumatoid arthritis, and asthma. The enzyme LTA(4) hydrolase represents an attractive target for pharmacological intervention in these disease states, since the action of this enzyme is the rate-limiting step in the production of LTB(4). Our previous efforts focused on the exploration of a series of analogues related to screening hit SC-22716 (1, 1-[2-(4-phenylphenoxy)ethyl]pyrrolidine) and resulted in the identification of potent, orally active inhibitors such as 2. Additional structure-activity relationship studies around this structural class resulted in the identification of a series of alpha-, beta-, and gamma-amino acid analogues that are potent inhibitors of the LTA(4) hydrolase enzyme and demonstrated good oral activity in a mouse ex vivo whole blood LTB(4) production assay. The efforts leading to the identification of clinical candidate SC-57461A (8d, 3-[methyl[3-[4-(phenylmethyl)phenoxy]propyl]amino]propanoic acid) are described.


Subject(s)
Enzyme Inhibitors/chemical synthesis , Epoxide Hydrolases/antagonists & inhibitors , beta-Alanine/chemical synthesis , Administration, Oral , Animals , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Leukotriene A4/biosynthesis , Leukotriene A4/blood , Mice , Structure-Activity Relationship , beta-Alanine/analogs & derivatives , beta-Alanine/chemistry , beta-Alanine/pharmacology
5.
J Biol Chem ; 272(16): 10601-7, 1997 Apr 18.
Article in English | MEDLINE | ID: mdl-9099707

ABSTRACT

Activated neutrophils release a variety of eicosanoids into the extracellular medium including arachidonic acid, 5-hydroxyicosatetraenoic acid, and leukotriene A4 and B4. In this study, the mechanism of arachidonic acid export has been examined using inside-out plasma membrane vesicles from pig polymorphonuclear leukocytes. Tritiated arachidonic acid associated rapidly with the membrane vesicles and crossed the membrane into the intravesicular space in a time-dependent and saturable manner. Half the maximal influx rate was measured at an arachidonate concentration of 5.7 microM, and a maximal influx velocity of 3.0 nmol/mg x min was determined at pH 6.8. Influx into vesicles was sensitive to a number of common anion transport inhibitors including pentachlorophenol, phloretin, diiodosalicylic acid, and quercetin as well as to the proteases trypsin and Pronase, suggesting a protein-dependent process. Furthermore, influx was temperature-sensitive with an energy of activation of 11.6 kcal/mol. Varying extravesicular concentration of ATP, Na+, or K+ had no impact on arachidonate influx, whereas changes in pH had a profound effect; optimum transport activity was observed at an extravesicular pH of 6, whereas raising the pH to 9.5 essentially abolished uptake. These results indicate and initially characterize a novel protein-facilitated arachidonate export mechanism in pig neutrophils.


Subject(s)
Arachidonic Acid/blood , Neutrophils/metabolism , Phagosomes/metabolism , Adenosine Triphosphate/pharmacology , Animals , Calorimetry , Cell Fractionation , Ethylmaleimide/pharmacology , Hydroxyeicosatetraenoic Acids/blood , In Vitro Techniques , Kinetics , Leukotriene A4/blood , Leukotriene B4/blood , Neutrophils/drug effects , Neutrophils/ultrastructure , Phagocytosis , Phagosomes/ultrastructure , Phloretin/pharmacology , Potassium/pharmacology , Quercetin/pharmacology , Sodium/pharmacology , Swine , Temperature
6.
J Lipid Res ; 38(4): 627-33, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9144078

ABSTRACT

The objective of this study was to determine the transcellular metabolism of leukotriene A4 by rabbit blood cells. mixed peripheral blood leukocyte preparations with and without platelets in a ratio of 1:40 were challenged with the Ca(2+)-ionophore A23187. 5-Lipoxygenase metabolites production was assessed by RP-HPLC coupled with diode-array UV detection. In light of the observation that leukotriene C4 production in leukocyte-platelet coincubation was the same as with leukocytes alone, mixed coincubation of human and rabbit blood cells was tested. Rabbit leukocytes with human platelets resulted in a significant increase of leukotriene C4 production, while no changes were observed in human leukocytes with or without rabbit platelets. In agreement with these results, intact rabbit platelets or rabbit platelet lysates, unlike human platelets, were not able to convert synthetic leukotriene A4 free acid to leukotriene C4. These data provide evidence that rabbit leukocytes are able to make a significant amount of leukotriene A4 available for transcellular metabolism, while rabbit platelets, unlike human platelets, lack leukotriene C4-synthase activity.


Subject(s)
Arachidonate 5-Lipoxygenase/metabolism , Blood Platelets/enzymology , Leukocytes/metabolism , Leukotriene A4/metabolism , Animals , Blood Platelets/drug effects , Blood Platelets/metabolism , Calcimycin/pharmacology , Cells, Cultured , Chromatography, High Pressure Liquid , Coculture Techniques , Glutathione Transferase/metabolism , Humans , Ionophores/pharmacology , Leukocytes/drug effects , Leukotriene A4/analysis , Leukotriene A4/blood , Rabbits , Spectrophotometry, Ultraviolet
7.
Biochem J ; 328 ( Pt 1): 225-9, 1997 Nov 15.
Article in English | MEDLINE | ID: mdl-9359857

ABSTRACT

We studied the metabolism of 14,15-dehydro-leukotriene A4 (14, 15-dehydro-LTA4) by human platelet leukotriene C4 (LTC4) synthase and polymorphonuclear leucocyte (PMNL) leukotriene A4 (LTA4) hydrolase. Metabolites were separated and identified using reversed-phase HPLC coupled to diode-array UV detection. Human platelets metabolize 14,15-dehydro-LTA4 to 14,15-dehydro-LTC4 with apparent kinetics identical with authentic LTA4. Metabolism to 14, 15-dehydro-LTC4 is inhibited by MK-886, a reported LTC4 synthase inhibitor in human platelets, with a potency comparable with that shown by LTA4. In contrast, neither human red-blood-cell lysates nor human PMNL enzymically convert 14,15-dehydro-LTA4 into 14, 15-dehydro-leukotriene B4. Minor amounts of 14,15-dehydro-LTC4, observed in some PMNL preparations, result from variable eosinophil contamination, as confirmed using highly purified neutrophil and eosinophil-enriched preparations. In addition, 14,15-dehydro-LTA4 irreversibly inhibits PMNL LTA4 hydrolase with an IC50 of 0.73 microM. The geometry of the methyl terminus of LTA4 does not influence the metabolism by human platelet LTC4 synthase. The double bond at C-14,15 is essential for the catalytic activity of LTA4 hydrolase but not for binding to this enzyme.


Subject(s)
Glutathione Transferase/metabolism , Leukotriene A4/analogs & derivatives , Leukotriene A4/metabolism , Binding Sites/drug effects , Blood Platelets/enzymology , Chromatography, High Pressure Liquid , Epoxide Hydrolases/antagonists & inhibitors , Erythrocytes/enzymology , Glutathione Transferase/blood , Glutathione Transferase/drug effects , Humans , Indoles/pharmacology , Leukotriene A4/blood , Lipoxygenase Inhibitors/pharmacology , Neutrophils/enzymology , Substrate Specificity/drug effects , Thiophenes/pharmacology
8.
J Immunol Methods ; 175(1): 97-105, 1994 Sep 30.
Article in English | MEDLINE | ID: mdl-7930643

ABSTRACT

An enzyme immunometric assay of LTC4 named SPIE-IA is described. The assay involves different sequential steps: (1) immunocapture of LTC4 by monoclonal anti-LTC4 antibodies coated on 96-well microtiter plates; (2) cross-linking of LTC4 via its amino group to the wells using glutaraldehyde; (3) treatment with HCl; (4) measurement of linked LTC4 using the same monoclonal anti-LTC4 antibodies labeled with acetylcholinesterase. A minimal detectable concentration of 2 pg/ml after 60 min of enzymatic reaction was obtained. Cross-reactivity was less than 15% with LTD4 or LTE4. The coefficient of variation was less than 6% in the 20-1000 pg/ml range. Good correlation was observed between SPIE-IA and a competitive enzyme immunoassay for biological samples. The different sequential steps of the assay are investigated.


Subject(s)
Immunoenzyme Techniques , Leukotriene C4/analysis , Acetylcholinesterase , Antibodies, Monoclonal , Blood Platelets/metabolism , Cells, Cultured , Chromatography, High Pressure Liquid , Haptens/immunology , Humans , Leukotriene A4/blood , Leukotriene C4/immunology , Sensitivity and Specificity
9.
Pediatr Res ; 35(3): 307-10, 1994 Mar.
Article in English | MEDLINE | ID: mdl-8190517

ABSTRACT

Glutathione synthetase deficiency (GSD) is an inborn error of glutathione (GSH) metabolism leading to a generalized intracellular GSH deficiency. Because GSH is required for leukotriene C4 (LTC4) synthesis, we studied synthesis and metabolism of several lipoxygenase products in two patients with GSD by radio-HPLC, UV spectrophotometry, and enzyme immunoassays. In both patients, LTC4 synthesis was significantly decreased in calcium ionophore-stimulated neutrophils (up to 0.4 ng/10(6) cells; controls, 5.0 +/- 0.9) and monocytes (up to 3.6 ng/10(6) cells; controls, 30.2 +/- 3.3). LTB4 synthesis was about seven times higher in GSD cells compared with controls, whereas synthesis of other 5-, 12-, and 15-lipoxygenase products and prostaglandin E2 was not affected. Neutrophils and monocytes from both patients showed a marked reduction in capacity to form [3H]LTC4 from [3H]LTA4 (9-14% of control values). Urinary LTE4 was finally found to be 50-fold lower in GSD, reflecting a decreased synthesis of cysteinyl LT in vivo. GSD may serve as a unique model for the linkage between LT synthesis and GSH metabolism in vivo.


Subject(s)
Glutathione Synthase/deficiency , Lipoxygenase/metabolism , Calcimycin/pharmacology , Child, Preschool , Erythrocytes/metabolism , Female , Glutathione/blood , Humans , In Vitro Techniques , Infant , Leukotriene A4/blood , Leukotriene C4/blood , Leukotriene E4/urine , Male , Monocytes/drug effects , Monocytes/metabolism , Neutrophils/drug effects , Neutrophils/metabolism , Sulfhydryl Compounds/blood
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