leukotriene-b4 and Mast-Cell-Sarcoma

The ability of linetastine (TMK688, 1-[¿5'-(3"-methoxy-4"-ethoxycarbonyloxyphenyl)-2',4'-pentadieno yl¿ aminoethyl]-4-diphenylmethoxypiperidine, CAS 110501-66-1) to inhibit leukotriene production and to antagonize the effect of histamine was examined in comparison with the ability of azelastine, an antiallergic drug having an antihistamine activity. Linetastine and its active metabolite TMK777 (1-[¿5'-(3"-methoxy-4"-hydroxyphenyl)-2',4'-pentadienoyl¿ aminoethyl]-4-diphenylmethoxypiperidine, CAS 101619-11-8) inhibited the release of leukotrienes B4 and C4 from calcium ionophore-stimulated human leukocytes. The respective IC50 values of leukotriene B4 were 1.2 x 10(-7) mol/l and 8.6 x 10(-8) mol/l, and those of leukotriene C4 were 1.5 x 10(-7) mol/l and 7.1 x 10(-8) mol/l. Azelastine also inhibited the release of leukotriene B4 and C4, but its IC50 values were higher than 1 x 10(-5) mol/l. Linetastine at 1-10 mg/kg p.o. inhibited the increase in leukotriene B4 and C4 production in the lungs during late asthmatic responses in actively sensitized guinea-pigs. The effect of 3.2 mg/kg lasted for more than 16 b. Since repeated oral administration of linetastine, 1 mg/kg once a day for 7 successive days, showed the same inhibitory effect on the increase in respiratory resistance and the leukotriene production as single oral administration, the effect of linetastine was neither tachyphylactic nor cumulative. Azelastine at 10 mg/kg had no effect on the leukotriene production. Linetastine TMK777 and azelastine dose-dependently inhibited the histamine-induced contraction of isolated guinea-pig trachea in a noncompetitive manner, the respective pD2 values were 7.28, 7.98 and 8.07. Linetastine inhibited histamine-induced bronchoconstriction dose-dependently at 1-10 mg/kg (p.o.) in guinea-pigs, and the effect lasted for more than 24 h. Repeated oral administration of linetastine, 0.32 to 3.2 mg/kg once a day for 7 successive days inhibited the histamine-induced bronchoconstriction, the same as single oral dosing. Azelastine at 0.32 mg/kg p.o. also showed antihistamine activity. In conclusion, linetastine inhibits both the production of leukotrienes and the effect of histamine at almost the same dose and the effects were long lasting.

Topics: Animals; Arachidonate 5-Lipoxygenase; Asthma; Bronchoconstriction; Female; Guinea Pigs; Histamine Antagonists; Humans; In Vitro Techniques; Leukocytes; Leukotriene B4; Leukotriene C4; Lipoxygenase Inhibitors; Lung; Male; Mammary Neoplasms, Experimental; Mast-Cell Sarcoma; Mice; Piperidines; Stimulation, Chemical; Trachea; Tumor Cells, Cultured

The effects of alcohols on the formation of leukotrienes, 5-HETE and prostaglandin D2 in mastocytoma cells and human neutrophils were studied. In murine mastocytoma cells, alcohols appear to have at least two different effects on the production of these arachidonic acid metabolites. At low levels of cellular arachidonic acid achieved after stimulation with calcium ionophore A23187 or addition of low levels of exogenous arachidonic acid, alcohols appear to have a general inhibitory effect on the production of lipoxygenase metabolites. In the presence of higher concentrations of cellular arachidonic acid, ethanol and methanol stimulated the production of lipoxygenase metabolites, but had no large stimulatory effect on the cyclo-oxygenase metabolite, prostaglandin D2. Under these conditions, n-propanol and t-butanol have inhibitory effects on leukotriene production. Human neutrophils are less sensitive to ethanol than mastocytoma cells, but stimulatory effects were still found at high ethanol concentrations (220-430 mM).

Topics: Alcohols; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Line; Cell-Free System; Chromatography, High Pressure Liquid; Ethanol; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase; Mast-Cell Sarcoma; Methanol; Mice; Neutrophils; Prostaglandin D2; Prostaglandins D; Time Factors

.eukotrienes are significantly involved in immunoregulation and in a variety of diseases, including asthma, inflammation and various allergic conditions. They are initially biosynthesized by 5-lipoxygenase from arachidonic acid, which can also be metabolized to prostaglandin endoperoxide by cyclooxygenase. The specific inhibitors for 5-lipoxygenase would be useful not only as tools for investigating the regulation mechanism of leukotriene biosynthesis, but also as drugs for clinical use. Although recently a few selective inhibitors have been reported, most of them are difficult to obtain, since they are new compounds. We found that caffeic acid, which is one of the most common reagents, is a selective inhibitor for 5-lipoxygenase and therefore for leukotriene biosynthesis. The inhibitory effect of its methyl ester on 5-lipoxygenase (ID50 = 4.8 X 10(-7) M) was stronger than that of caffeic acid itself (ID50 = 3.7 X 10(-6) M). Caffeic acid inhibited 5-lipoxygenase in a non-competitive manner. Caffeic acid and its methyl ester did not inhibit prostaglandin synthase activity at all, at least up to 5 X 10(-4) M, but rather stimulate at higher doses. The biosynthesis of leukotriene C4 and D4 in mouse mast tumor cells was also inhibited completely with 10(-4) caffeic acid. Besides, caffeic acid had little effect on arachidonic acid metabolism in platelet at less than 1 X 10(-5) M, but at higher doses it showed a definite inhibitory effect, i.e., thromboxane B2, HHT (12(S)-hydroxy-5,8,10-heptadecatetraenoic acid) and 12-HETE (12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid) syntheses were inhibited 33, 40 and 80% at 1 X 10(-4) M, respectively. Platelet aggregation induced by arachidonic acid was also inhibited by caffeic acid at high dose, while platelet aggregation induced by ADP is not influenced by caffeic acid at all. The observations on caffeic acid and its derivatives may contribute to leukotriene research.

Topics: Animals; Arachidonate Lipoxygenases; Blood Platelets; Caffeic Acids; Cinnamates; Cyclooxygenase Inhibitors; Leukotriene B4; Lipoxygenase Inhibitors; Mast-Cell Sarcoma; Rats; SRS-A

Isolated dog mastocytoma cells sensitized with dog anti-ragweed IgE and challenged with ragweed antigen or incubated with ionophore A23187 or the carboxy-terminal dodecapeptide of platelet factor 4, PF4(59-70), release histamine and concurrently generate leukotrienes B4, C4, and D4. In contrast, the exposure of mastocytoma cells to 0.1-3 micrograms/ml of 15-hydroxyeicosatetraenoic acid (15-HETE) stimulates selectively the generation of leukotrienes, in the absence of histamine release, while 0.1-1 micrograms/ml of compound 48/80 releases histamine without enhancing the generation of leukotrienes. That natural stimuli are capable of selectively activating one synthetic or secretory compartment of mast cells suggests that separate subsets of receptors as well as different biochemical events may serve to mobilize each class of mediators.

Topics: Allergens; Animals; Arachidonic Acids; Calcimycin; Cell Transformation, Neoplastic; Dogs; Histamine Release; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Mast-Cell Sarcoma; p-Methoxy-N-methylphenethylamine; Platelet Factor 4; SRS-A

Isolated dog mastocytoma cells sensitized with dog anti-ragweed IgE and challenged with ragweed antigen generate the C-6 peptide leukotriene (LT) constituents of the slow-reacting substance of anaphylaxis (SRS-A), LTC4 and LTD4, and the potent leukocyte chemotactic factor, LTB4, as well as 15-hydroxyeicosatetraenoic acid (15-HETE), 12-HETE, and 5-HETE. Antigen challenge evoked a mean maximum production of LTC4, LTD4, and LTB4, respectively, of 16.3 ng, 4.6 ng, and 6.7 ng per 10(6) mastocytoma cells within 5 min at 37 degrees C, which was maintained for up to 45 min. The maximum production of leukotrienes by mastocytoma cells activated with optimum concentrations of 0.2 to 1 microM calcium ionophore A23187 was 35% to 50% greater than that achieved by antigen challenge, without alterations in the relative quantities of leukotrienes, but was realized only after 30 min at 37 degrees C. The leukotriene products of mastocytoma cells were identified by high-performance liquid chromatography, spectral properties, and immunoreactivity with mono-specific antisera, and exhibited the same concentration-dependence of biologic activity as authentic synthetic standards. The ratio of quantities of C-6 peptide leukotrienes generated was attributable in part to the rate of peptidolytic conversion of LTC4 to LTD4 in the mastocytoma cells, which was 33% per 30 min at 37 degrees C. The rapid maximum response to IgE-dependent stimulation and the unique spectrum of products distinguishes the secretion of leukotrienes by dog mastocytoma cells from that by basophils and some other types of mast cells and suggests diverse contributions of mast cell leukotrienes to immediate and late hypersensitivity reactions.

Topics: Animals; Anti-Bacterial Agents; Arachidonic Acid; Arachidonic Acids; Calcimycin; Dogs; Immunoglobulin E; Leukotriene B4; Mast-Cell Sarcoma; Pollen; SRS-A; Time Factors

The present studies were undertaken to examine the hypothesis that ethanol could affect cellular biosynthesis in the murine mastocytoma cell of prostaglandins and leukotrienes, oxidative metabolites of arachidonic acid, at concentrations that could be encountered in vivo as well as during in vitro experiments. The effects of ethanol which encompass these concentration ranges (200-1000 mg%) can be summarized as follows: first in the absence of exogenous arachidonic acid, ethanol caused a dose dependent decrease in the production of leukotrienes which was statistically significant at 200 mg%. At 1000 mg%, ethanol caused a 20-50% decrease in leukotrienes and a 21% decrease in the amount of prostaglandin D2 (PGD2) formed in these cells. Secondly, when cells were incubated with exogenous arachidonic acid (14 micrograms/ml), large increases in both PGD2 and leukotrienes occurred. Under these conditions, ethanol caused a further increase in the amount of leukotrienes and a small increase in the amount of PGD2 formed. This stimulatory effect was specific for ethanol since neither t-butanol nor n-butanol caused the enhanced production of leukotrienes with exogenous arachidonic acid. Thus, these experiments suggest that ethanol affects metabolism of arachidonic acid at reasonably low doses (200-400 mg%) of ethanol in a manner dependent on the free arachidonic acid in the tissue. Also, in vitro experiments in which ethanol is used as a solvent for arachidonic acid could be greatly affected by high levels of ethanol (500-1000 mg%) which are frequently utilized.

Topics: 1-Butanol; Animals; Arachidonic Acid; Arachidonic Acids; Butanols; Calcimycin; Chromatography, High Pressure Liquid; Ethanol; Leukotriene B4; Mast-Cell Sarcoma; Mice; Prostaglandin D2; Prostaglandins D; SRS-A

Topics: Animals; Arachidonic Acids; Diethylcarbamazine; Female; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Mast-Cell Sarcoma; Mice; Prostaglandins; Sarcoma, Experimental; SRS-A

Topics: Animals; Cells, Cultured; Chromatography, High Pressure Liquid; Female; Gas Chromatography-Mass Spectrometry; Leukotriene B4; Mast-Cell Sarcoma; Mice; Sarcoma, Experimental; Spectrophotometry, Ultraviolet; SRS-A

Previous methods for the recovery and quantitation of leukotrienes have involved tedious extraction procedures, and high-performance liquid chromatographic (HPLC) techniques with significant limitations. We have designed a method to extract leukotrienes from biologic fluids using commercially available silica mini-columns requiring minimal preparation. Sample clarification is followed by a sensitive and reproducible HPLC technique which separates and quantifies the leukotrienes LTC4, LTD4, LTB4 (and at least three of their isomers). The entire procedure requires less than one hour per sample.

Topics: Cell Line; Chromatography, High Pressure Liquid; Humans; Isomerism; Leukotriene B4; Mast-Cell Sarcoma; SRS-A

A neoplastic mast cell tumor was grown in mice which had been raised since birth on a diet enriched with eicosapentaenoic acid. Intact harvested mastocytoma cells were stimulated with calcium ionophore A23187 to produce lipoxygenase products from the polyunsaturated fatty acids liberated from the cellular membranes. Leukotriene B4, B5, C4, and C5 were isolated and characterized by HPLC retention time, ultraviolet absorption spectrometry and mass spectrometry. The arachidonic acid content of the mast cell tumor lipids was altered from 9.2 to 3.9 mole % while eicosapentaenoic acid increased from 0.5 to 4.5 mole % in response to the fish oil-supplemented diet. The relative amounts of arachidonic and eicosapentaenoic acids (3.9 and 4.5 mole % respectively) were associated with similar amounts of LTB4 and LTB5 synthesized by the cells. These results suggest that the epoxide leukotriene (LIA) derivative can be made efficiently from either arachidonic or eicosapentaenoic acids when both are present in cellular lipids. In contrast, the ratio of LTC4 to LTC5 (10 to 1) indicates that the reaction of LTA with glutathione may be critically dependent upon the structure of the unsaturated fatty acid with the ratio of LTC4/LTB4 (2.0) more than 10 times greater than that (0.16) for LTC5/LTB5.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Autacoids; Calcimycin; Chromatography, High Pressure Liquid; Dietary Fats; Eicosanoic Acids; Eicosapentaenoic Acid; Fatty Acids, Unsaturated; Female; Fish Oils; Leukotriene B4; Mass Spectrometry; Mast-Cell Sarcoma; Mice; Pregnancy; Spectrophotometry, Ultraviolet; SRS-A

Topics: Animals; Arachidonic Acids; Chromatography, High Pressure Liquid; Leukotriene B4; Mast-Cell Sarcoma; Mice; Neoplasms, Experimental; Prostaglandins B; Solvents; SRS-A