Vascular smooth muscle cell leukotriene C4 synthesis: requirement for transcellular leukotriene A4 metabolism (original) (raw)
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European Journal of Biochemistry, 1988
Incubation of human endothelial cells with leukotriene A4 resulted in the formation of leukotrienes B4, C4, D4 and E4. Endothelial cells did not produce leukotrienes after stimulation with the ionophore A23187 and/or exogenously added arachidonic acid. However, incubation of polymorphonuclear leukocytes with ionophore A231 87 together with endothelial cells led to an increased synthesis of cysteinyl-containing leukotrienes (364%, mean, n = 11) and leukotriene B4 (52%) as compared to leukocytes alone. Thus, the major part of leukotriene C4 recovered in mixed cultures was attributable to the presence of endothelial cells. Similar incubations of leukocytes with fibroblasts or smooth muscle cells did not cause an increased formation of leukotriene C4 or leukotriene B4.
Endothelial cells can synthesize leukotriene B
Journal of Vascular Surgery, 1990
Leukotriene B4 (LTB,) from vascular endothelium may play a key role in the genesis of atherosclerotic lesions. However, the ability of this tissue to synthesize LTB~ is controversial. To resolve this issue arachidonic acid metabolism was characterized in cultures of confluent monolayers of a rabbit aortic endothelial cell line by use of both high-pressure liquid chromatography and radioimmunoassay. Cells were grown to confluence in Dulbecco's modified Eagle's medium/Ham's F12 with 5% fetal bovine serum. Lipoxygenase activity was studied by placing the cells in Hank's balanced salt solution with 2 wmol/L indomethacin. After 30 minutes preincubation with indomethacin cells were exposed to either arachidonic acid (10 ~mol/L) or arachidonic acid labeled with radioactive carbon (14C) (1 ~tCi; SA 58 mCi/mmol) and then stimulated with 9.5 ~mol/L calcium ionophore A23187 for 55 minutes. Studies of the cyclooxygenase activity were performed without preincubating with indomethacin. Samples were prepared for high-pressure liquid chromatography by evaporation to dryness under a vacuum and resuspending in 2 ml of 1 : I methanol/water. Tritium-labeled standards were added before loading the ~4C-labeled samples on the column. Radiolabeled arachidouic acid metabolites were separated by high-pressure liquid chromatography and detected by means of a dual channel flowthrough radiodetector that monitors both ~4C and SH. Based on coelution with authentic standards three lipoxygenase metabolites of arachidonic acid have been identified: LTB~, 12-and 5-hydroxyeicosatetraneoic acid. Leukotriene B4 was further characterized by ultraviolet spectral analysis and inhibition studies with use of nordihydroguaiaretic acid. Quantitation was facilitated by commercially available radioimmunoassay kits. An average of 600 pg LTBJ 106 cells was measured from separate experiments. Negative controls produced no LTB4. These data demonstrate that rabbit aortic endothelial cells when stimulated with calcium ionophore in the presence of exogenous arachidouic acid synthesize LTB4, an inflammatory mediator that may play an important role in the pathogenesis of atheroslcerosis.
Specific binding of leukotriene C4 to endothelial cell membranes
Prostaglandins, Leukotrienes and Medicine, 1987
Vascular endothelium is a target for leukotriene C4 (LTC4) as demonstrated by previous in vivo and culture experiments. Binding assays were carried out at 0 degrees C on membrane fraction obtained from bovine aortic endothelial cells in culture. Specific binding sites (Kd = 49.9 +/- 6.3 nmol X 1(-1), N = 1.2 X 10(6) sites per cell) for LTC4 were demonstrated in this preparation. Competition studies showed that LTB4, LTD4 and LTE4 did not displace LTC4 from its binding sites. FPL 55712, a sulfidopeptide antagonist, was seen to be a weak competitor and reduced glutathione exhibited a significant affinity for the binding site. The possible receptor role of this site is discussed.
Journal of cell science, 1989
During the first few days in primary culture arterial smooth muscle cells (SMCs) go through a transition from a contractile to a synthetic phenotype. Morphologically, this process includes loss of myofilaments and formation of an extensive rough endoplasmic reticulum and a large Golgi complex. Functionally, it leads to the cells losing their contractility, beginning to secrete extracellular matrix components, and dividing in response to growth factor stimulation. Similar changes in the structure and function of the SMCs occur in the initial stages of atherogenesis. The object of the present investigation was to study the effects of leukotrienes on the differentiated properties and growth of rat aortic SMCs in primary culture. Enzymically isolated cells were seeded directly on a plastic surface in serum-containing medium or on a substratum of plasma fibronectin in serum-free medium. The change in cell morphology was followed by transmission electron microscopy, and the activation of ...
Leukotriene C4 biosynthesis in isolated August rat peritoneal leukocytes
Mediators of Inflammation, 1996
The mixed leukocyte population obtained from the peritoneum of the August rat is a potentially important experimental model of inherent eosinophilia that has not been well characterized. In the present study, isolated cell preparations generated a concentration-dependent release of leukotriene (LT) C4when exposed to the Ca2+ionophore A23187, reaching maximal stimulation at 5.0 μM. This response was inhibited by the 5-lipoxygenase activating protein antagonist MK-886 (0.1 μM), nominally Ca2+and Mg2+-free incubation media and by activation of protein kinase C via phorbol 12-myristate 13-acetate (50 nM). These findings establish a model system for investigating LTC4profiles contingent with innate peritoneal eosinophilia and are consistent with the hypothesis that cellular LTC4biosynthesis is phosphoregulated.
Ca2+ mobilization with leukotriene A4 and epoxytetraenes in human neutrophils
Biochemical Pharmacology, 1990
The biosynthesis of leukotrienes and lipoxins involves epoxide-containing intermediates which may be subject to several routes of transcellular metabolism. We have examined the capacity of leukotriene A4 (LTA,) and 15S-hydroxy-5,6-oxido-7,9,13-rrans-ll-ci-eicosatetraenoic acid [5(6)epoxytetraene] to stimulate the mobilization of free cytosolic calcium ([Ca'+li) in human blood neutrophils. To gain insight into structure-activity relationships, a putative intermediate in lipoxin biosynthesis, SS-hydroxy-14,15-oxido-6,10,12-truns-8-c~-eicosatetraenoic acid [14(15)-epoxytetraene], was prepared by total synthesis. When added to fura-loaded neutrophils, each of these compounds provoked a rapid and transient increase in [Caz+li ( maximum by 8 set) which returned to baseline within 60-90 sec. CaZ+ mobilization with LTAl was dose dependent and, at 1 PM, the efficacies of LTA4 and LTB4 were quantitatively similar. The 5(6)-epoxytetraene and 14(15)-epoxytetraene were less potent than LTA+ Prior exposure of the cells. to dthyieneglycolbis(aminoethyle~her)tetra-acetate (E'GTA) (60 sec. 3 mM) did not diminish either the amplitude or the extent of ICa2+1, elicited bv LTA,. Methvl Prostaglandins 30: 935-947, 1985. 29. Jones DB, Marante D, Williams BC and Edwards CRW, Adrenal synthesis of corticosterone in response to ACTH in rats is influenced by leukotriene A4 and by lipoxygenase intermediates. J Endocrinolll2: 253-258, 1987. 30. Beckman JK, Gay JC, Brash AR, Lukens JN and lets.
Journal of lipid research, 1997
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 mak...