And THP-1 cells do not release LTB4, LTC4, or LTD4 in response to A23187

U937 and THP-1 cells possess some characteristics of human mononuclear phagocytes, cells which synthesize and release LTB₄, LTC₄, and LTD₄. Incubation of these cells with recombinant human interferongamma (IFN-gamma) or Phorbol Myristate Acetate (PMA) induces a more differentiated cell state. We hypothesized that U937 and THP-1 cells would release LTB₄, LTC₄, and LTD₄ in response to stimulation with the non-physiologic agonist, calcium ionophore A23187 and that preincubation with IFN-gamma or PMA might alter leukotriene release by thes cells. We cultured both cell lines for 48 hours in the presence and absence of IFN-gamma (10000 units/ml)n and for 120 hours in the presence and absence of PMA (160 nM) and then challenged them with A23187 (5uM) for 30 minutes at 37°C. The supernatants were deproteinated and assayed by RIA for LTB₄ and LTC₄ and by RP-HPLC for LTB₄, LTC₄, and LTD₄. Neither U937 nor THP-1 cells released quantities of leukotrienes detectable by RIA, <0.3ng/5 × 10⁶ cells. Peripheral blood mononuclear phagocytes from normal volumteers, cultured and challenged in vitro at under identical conditions, released 11.3 ± 2.9 ng LTB₄ and 2.0 ± 1.5 ng LTC₄/10⁶ viable monocytes. The lack of leukotriene production by U937 and THP-1 cells was not altered by preincubation for 48 hours with IFN-gamma (n=3) nor by preincubation with PMA for 120 hours (n=3). We conclude 1) U937 and THP-1 cells do not appear to be appropriate in vitro models for the examination of leukotriene release from normal mononuclear phagocytes. 2) Pre-incubation of U937 and THP-1 cells with IFN-gamma or PMA under the conditions tested, does not induce the ability of these cell lines to release leukotrienes.     

Specificity of receptors for leukotrienes A4, B4, C4, D4, E4 and histamine on the guinea-pig lung parenchyma. Effect of FPL-55712 and desensitization of the myotropic activity

The novel metabolites of arachidonic acid, leukotriene (LT) A₄, B₄, C₄, D₄ and E₄ have potent myotropic activity on guinea-pig lung parenchymal strip . The receptors responsible for their action were characterized using desensitization experiments and the selective SRS-A antagonist, FPL-55712. During the continuous infusion of LTB₄, the tissues became desensitized to LTB₄ but were still responsive to histamine, LTA₄, LTC₄, LTD₄ and LTE₄. When LTD₄ was infused continuously, the lung strips contracted to LTB₄ and histamine but were no longer responsive to LTA₄, LTC₄, LTD₄ and LTE₄. Furthermore, FPL-55712 (10 ng ml⁻¹− 10 ug ml⁻¹) produced dose-dependent inhibitions of LTA₄, LTC₄, LTD₄ and LTE₄ without inhibiting the contraction to LTB₄ and histamine. On the basis of these results, it appears that the guinea-pig lung parenchyma may have one type of receptor for LTB₄ and another for LTD₄; LTA₄, LTC₄ and LTE₄ probably act on the LTD₄ receptor.     

Questionable role of leukotriene B4 in monosodium urate (MSU)-induced synovitis in the dog

Monosodium urate (MSU)-induced synovitis in the dog's stifle (knee joint) is similar to an acute gouty attack in man in which a loss of function of the joint correlates with massive influx of neutrophils and the release of an assortment of inflammatory mediators (e.g. histamine, bradykinin, lysosomal enzymes, complement and eicosanoids) into the synovial space. We found in the urate-induced inflammatory exudates 3 hr post MSU the following: 88 million leukocytes/ml (95% neutrophils) and eicosanoid concentrations of LTB₄, LTC₄, and PGE₂ of < 0.1, 1.4 and 20 ng/ml, respectively. Isotonic saline injected knee joints at 3 hr contained 5 million leukocytes/ml (95% neutrophils) and concentrations of LTB₄, LTC₄, and PGE₂ of < 0.1, 0.7 and 0.2 ng/ml, respectively. Intrasynovial injections of 1 μg LTB₄, 10 μg PGE₂ or the combination of LTB₄ and PGE₂ produced no reduction of paw pressure for up to 3 hr. Leukocyte concentrations measured at 3 hr in joints injected with these arachidonic acids metabolites were similar to saline controls. These results question the role of LTB₄ as a chemotactic and inflammatory mediator in urate-induced synovitis in the dog but confirm the importance of PGE₂ and possibly LTC₄ in this model.     

Leukotriene B4, polymorphonuclear leukocytes and inflammatory exudates in the rat

Leukocyte numbers and Leukotriene B₄- (LTB₄-) and LTC₄-immunoreactivity were measured in inflammatory exudates obtained from sponges impregnated with several irritants implanted subcutaneously in the rat. Sponges containig 1% uric acid, carragennan or zymosan were implanted for 5h and compared to saline sponges. Increases in leukocyte numbers and LTB₄-immunoreactivity were found in the presence of irritants, the highest concentrations being observed in the presence of zymosan. The presence of LTB₄ was confirmed by liquid chromatographic (HPLC) analysis. A time course study was carried out with zymosan-impregnated sponges and the maximal rate of leukocyte infiltrations was found to coincide with the maximal levels of LTB₄-immunoreactivity. The LTC₄-immunoreactivity was low and following analysis by HPLC was concluded to be unrelated to leukotrienes. The levels of LTB₄-immunoreactivity, but not the numbers of leukocytes, were elevated compared to corresponding controls in sponges containing 0.01% ionphore A23187 (untreated rats) or in sponges containing zymosan (rats pretreated with indomethacin; 3 and 10 mg/kg p.o.). Impregnation of sponges with 3 × 10⁻⁶M LTB₄ but not 3 × 10⁻⁷M LTB₄ induced a significant leukocyte migration. It was concluded that LTB₄ can induced leukocyte migration into sponge exudates in the rat but that measurements of LTB₄ in such exudates can not be correlated with the degree of leukocyte infiltration.     

Leukotriene D4 and E4 formation by plasma membrane bound enzymes

The homogenate of rat basophilic leukemia cells produces both the dihydroxy-leukotrienes and the peptido-leukotrienes (LT) C₄, D₄ and E₄. The enzymes responsible for the formation of LTA₄ and LTB₄ are in the soluble fraction while the enzymes for LTC₄, LTD₄ and LTE₄ are particulate (10, 000 × g pellet). Centrifugation of the 10, 000 × g pellet over a sucrose gradient resulted in two subfractions, a membrane fraction and a pellet (sucrose pellet.) The fractions were incubated with LTC₄, and the products were identified by bioassay, HPLC and UV spectra. The membrane fraction contained the enzymes γ-glutamyl transpeptidase and amino peptidase which convert LTC₄ to LTD₄ and LTD₄ to LTE₄, respectively. When incubated with LTC₄, the membrane fraction showed a dose dependent formation of LTD₄ and a time course which reached a plateau at 30 to 45 minutes. Addition of serine borate blocked the formation of LTD₄, and cysteine blocked LTE₄. We conclude that the γ-glutamyl transpeptidase and the amino peptidase which produce LTD₄ and LTE₄ respectively are plasma membrane bound.     

Inhibition by 16, 16-dimethyl PGE2 of ethanol-induced gastric mucosal damage and leukotriene B4 and C4 formation

The effects of PGE₂ and its stable analogue, 16, 16 dimethyl PGE₂ (dmPGE₂) were investigated on ethanol-induced gastric mucosal haemorrhagic lesions and leukotriene formation in the rat. Exposure of the rat gastric mucosa to ethanol , produced a concentration-related increase in the mucosal formation of leukotriene B₄ (LTB₄) which was correlated with macroscopically-apparent haemorrhagic damage to the mucosa. Challenge with absolute ethanol likewise enhanced the mucosal formation of LTC₄ whereas the mucosal formation of 6-keto-PGF_1α was unaffected. Challenge of the rat gastric mucosa with ethanol induced a concentration-dependent increase in the formation of LTB₄ and LTC₄, but not 6-keto PGF_1α. Pretreatment with PGE₂ (200–500μg/kg p.o.) prevented the haemorrhagic mucosal damage induced by oral administration of absolute ethanol but not the increased formation of leukotrienes by the mucosa. In contrast, pretreatment with a high dose of dmPGE₂ (20μg/kg p.o.) prevented both the gastric mucosal lesions and the increase mucosal leukotriene formation. The differences in the effects of these prostaglandins may be related to the nature or degree of protection of the gastric mucosa. Thus, high doses of dmPGE₂ but not PGE₂ may protect the cells close the luminal surface of the mucosa and hence reduce the stimulation of leukotriene synthesis by these cells.     

Species difference in increased vascular permeability by synthetic leukotriene C4 and D4

The activity of synthetic LTC₄ was tested in guinea-pig ileum and was 200 times more potent than histamine in contraction of the ileum (3 × 10^?11 M- 3 × 10^?9 M). The activities of LTC₄ and LTD₄ in increased vascular permeability in guinea pigs, rats and rabbits were compared with those histamine, bradykinin and prostaglandin (PG) E₂. LTC₄ was approximately equipotent to bradykinin on a molar basis in guinea pigs and rats and 5–100 times more potent than histamin. LTD₄ was about 10 times more potent than LTC₄ in guinea pigs and as equipotent to LTC₄ in rats. On the contrary, in rabbits, neither LTC₄ (upto 30 nmole/site) nor LTD₄ (1 nmole/site) induced the dye exduation. These results show that species difference is present in activity of LTC₄ and LTD₄ in vascular permeability. Furthermore, in guinea pigs, the vascular permeability increased by LTC₄ was not affected after pretreatment with pyrilamine (2.5 mg/kg, i.v.), and LTC₄ and LTD₄ did not potenciate the activity of bradykinin in vascular permeability.     

Pulmonary microcirculatory responses to leukotrienes B4, C4 and D4 in sheep

The pulmonary microvascular responses to leukotrienes B₄, C₄ and D₄ (total dosage of 4 μg/kg i.v.) were examined in acutely-prepared halothane anesthetized and awake sheep prepared with lung lymp fistulas. In anesthetized as well as unanesthetized sheep, LTB₄ caused a marked and transient decrease in the circulating leukocyte count. Pulmonary transvascular protein clearance (pulmonary lymph flow x lymph-to-plasma protein concentration ratio) increased transiently in awake sheep, suggesting a small increase in pulmonary vascular permeability. The mean pulmonary artery pressure (P ) also increased. In the acutely-prepared sheep, the LTB₄-induced pulmonary hemodynamic and lymph flow responses were damped. Leukotriene C₄ increased P to a greater extent in awake sheep than in anesthetized sheep, but did not significantly affect the pulmonary lymph flow rate (Q̇_lym) and lmph-to-plasma protein concentration (L/P) ration in either group. LTD₄ increased P and Q̇_lymp in both acute and awake sheep; Q̇_lym increased without a significant change in the L/P ratio. The LTD₄-induced rise in P occurred in association with an increase in plasma thromboxane B₂ (Txb₂) cocentration. The relativity small increase in Q̇_lym with LTD₄ suggests that the increase in the transvascular fluid filtration rate is the result of a rise in the pulmonary capillary hydrostatic pressure. In conclusion, LTB₄ induces a marked neutropenia, pulmonary hypertension, and may transiently increase lung vascular permeability. Both LTC₄ and LTD₄ cause a similar degree of pulmonary hypertension in awake sheep, but had different lymph flow responses which may be due to pulmonary vasoconstriction at different sites, i.e. greather pre-capillary constriction with LTC₄ because Q̇_lym did not change and greater post-capillary constriction with LTD₄ because Q̇ increased with the same rise in P .     

Generation of leukotriene B4 and leukotriene E4 from porcine pulmonary artery

When chopped porcine pulmonary arteries were incubated with calcium ionophore A23187 (1) in the presence of indomethacin there was a time dependent generation of a substance which produced contractions of superfused strips of guinea-pig ileum smooth muscle (GPISM) which were indistinguishable from those induced by LTD₄. This material however had a different retention time from LTD₄ when subjected to HPLC and co-chromatographed with synthetic LTE₄. In addition to LTE₄ a substance which had properties indistinguisable from those of LTB₄ when assayed on a combination of guinea-pig lung parenchymal strips (GPP) and GPISM (2) was generated from the pulmonary artery. This substance co-chromatographed with synthetic LTB₄. The adventitia and intima were the richest source of LTE₄, the adventitia releasing slightly more than the intima. The output of LTB₄ and LTE₄ was inhibited by 6,9-deepoxy-6,9-(phenylimino)-Δ^6,8 prostaglandin I₁ (U-60,257). Nordihydroguaiaretic acid (NDGA) inhibited the generation of LTE₄.     

Detection of leukotriene C4 and D4 in the exudate of rat carrageenin-induced pleurisy

Rat carrageenin-induced pleurisy was used as an acute exudative inflammatory model. The crude ethanol extract of the pleural fluid at 5 hr after carrageenin injection caused the very slow contraction of guinea-pig ileum, which was antagonized by FPL 55712 (1 μg/ml). The ethanol extract was cleaned by LH-20 and was rendered for separation of LTC₄ and LTD₄ by reversed-phase high-performance liquid chromatography (HPLC). Two peaks which showed the same retention time on HPLC as those of LTC₄ and LTD₄ had the contractile activity of guinea-pig ileum and the ratios of the contractile activity to the height on HPLC agreed with those of synthetic LTC₄ and LTD₄. Two peaks of Δ⁶-trans-LTB₄, 5S,12R-(E,E,E,Z)-diHETE and 5S, 12S-(E,E,E,Z)-diHETE, were detected, but the appreciable amount of LTB₄ was smaller than that of each Δ⁶-trans-LTB₄ in the pleural fluid at 5 hr.     

Functional expression of single-chain antibody to leukotriene C4

Leukotriene C₄ (LTC₄) is synthesized by binding of glutathione to LTA₄, an epoxide derived from arachidonic acid, and further metabolized to LTD₄ and LTE₄. We previously prepared a monoclonal antibody with a high affinity and specificity to LTC₄. To explore the structure of the antigen-binding site of a monoclonal antibody against LTC₄ (mAbLTC), we isolated full-length cDNAs for heavy and light chains of mAbLTC. The heavy and light chains consisted of 461 and 238 amino acids including a signal peptide with molecular weights of 51,089 and 26,340, respectively. An expression plasmid encoding a single-chain antibody comprising variable regions of mAbLTC heavy and light chains (scFvLTC) was constructed and expressed in COS-7 cells. The recombinant scFvLTC showed a high affinity with LTC₄ comparable to mAbLTC. The scFvLTC also bound to LTD₄ and LTE₄ with 48% and 17% reactivities, respectively, as compared with LTC₄ binding, whereas the antibody showed almost no affinity for LTB₄.     

Retinoid-induced inhibition of bosinophil LTC4 production

Naturally occuring and synthetic retinoids demonstrate a marked antiinflammatory effect when employed in such disorders as acne and psoriastis. This effect may result in part from their inhibition of release of potent mediators (e.g. eicosanoids) by inflammatory cells. In this study, we examined the effect of eight retinoids (tretinoin, isotretinoin, retinol, retinal, acitretin, retinyl palmitate, etretinate, Ro 15–0778) on the release of leukotriene (LT)C₄, an important lipid mediator generated by eosinophils. Tretinoin, isotretinoin, retinol, retinal, and acitretin at 10⁻⁵ M or 10⁻⁴ M concentrations inhibited LTC₄ release by A23187-stimulated horse eonsinophils in vitro; 10⁻⁴ M retinyl palmitate was also inhibitory. However, 10⁻⁵ M etretinate augmented A23187-induced LTC₄ release, and the arotinoid Ro 15–0778 had no effect on LTC₄ production. These data suggest that selected retinoids may have potential use in the reduction of LTC₄ generation by eosinophils. This inhibition could be beneficial in the theraphy of such diseases as bronchial asthma in which release of LTC₄ may be involved in the inflammtory process.     

Pharmacological comparison of L-serine borate and glutathione as inhibitors of metabolism of LTC4 to LTD4 by the isolated guinea pig trachea

Cumulative dose-response curyes to leukotriene C₄ (LTC₄) and leukotriene D₄ (LTD)₄ were obtained on indomethacin (5 μM) treated isolated guinea pig tracheal spiral strips. LTC₄ curves, in the presence of either glutathione (GSH; 10 mM) or L-serine borate (SB; 45 mM), were not antagonized by FPL-55712 (3 μM), a selective LTD₄ receptor antagonist. LTC₄ curves on trachea treated with a lower concentration of GSH (1 mM), and LTD₄ curves were competitively antagonized by FPL-55712. LTC, curves on GSH (10 mM) treated trachea were 2 fold to the left of those on SB treated tissues. This effect of GSH was blocked by pretreatment with nordihydro-guiaretic acid (30 μM), an inhibitor of 5-lipoxygenase.GSH (10 μM) and SB (45 mM) are effective inhibitors of conversion of LTC₄ into functionally important levels of LTD₄ by the guinea pig trachea. In addition, GSH appeares to enhance LTC₄ responsiveness by increasing synthesis of a contractile 5-lipoxygenase product(s), possibly LTC₄. From the data it is suggested that for inhibition of LTC₄ metabolism, SB may be more usefull when examining responses to exogenously applied LTC₄, while GSH (10 mM) may be useful when examining responses to endogenously generated LTC₄.     

Vasoconstrictor effects of leukotrienes C4 and D4 in the feline mesenteric vascular bed

The effects of leukotriene C₄ (LTC₄) and leukotriene D₄ (LTD₄) in the feline mesenteric vascular bed were investigated under conditions of controlled blood flow so that changes in perfusion pressure directly reflect changes in vascular resistance. Intra-arterial injections of LTC₄ and LTD₄ (0.3–3.0 μg) increased perfusion pressure in a dose-related fashion. Vasoconstrictor responses to LTC₄ and LTD₄ were similar to norepinephrine (NE) whereas mesenteric vasoconstrictor response to the thromboxane analog, U46619, was markedly greater than were responses to LTC₄ and LTD₄. Meclofenamate in a dose that greatly attenuated the systemic depressor response to arachidonic acid was without effect on vasoconstrictor responses to LTC₄ and LTD₄, NE and U46619 in the mesenteric vascular bed. The present data show that LTC₄ and LTD₄ possess significant vasoconstrictor activity in the feline mesenteric vascular bed. In addition, the present data suggest that products of the cyclooxygenase pathway do not mediate vasoconstrictor responses to LTC₄ and LTD₄ in the intestinal circulation of the cat.     

Bio-synthesis of PGD2 and TXB2 by rabbit blood monocytes

A method for the preparation of a highly purified sample of rabbit blood monocytes is described. The metabolism of arachidonic acid (AA) in these cells was studied. Mononuclear cells were prepared by centrifugation on Ficoll-Paque gradients and the monocytes were obtained by further centrifugation and adherence onto plastic culture dishes. These procedures provided a preparation which contained 95% monocytes (non-specific esterase positive). Incubation of [1-¹⁴C]-AA with these cells produced four major metabolites which were separated by TLC; these corresponded to prostaglandin (PG) D₂, thromboxane (TX) B₂, 12-hydroxyheptadecatrienoic acid (HHT) and 12-/15- hydroxyeicosatetraenoic acid (HETE). A minor product which co-migrated with PGE₂ was also detected but neither 6-keto-PGF_1α nor PGF_2α were detected. Also, there was no evidence of the formation of 5-lipoxygenase products (5-HETE and LTB₄) by rabbit monocytes with or without calcium-ionophore A23187-stimulation. The production of PGD₂, TXB₂ and PGE₂ was further confirmed by analyzing [³H]-AA metabolites using high-performance liquid chromatography (HPLC) with tritiated standards as references. The biosynthesis of these compounds from endogenous substrate in A23187-stimulated monocytes was confirmed by specific radioimmunoassays with or without prior HPLC separation. The synthesis of immunoreactive LTB₄ and LTC₄ by A23187-stimulated cells was also monitored and found to be relatively low. The synthesis of PGD₂, TXB₂ and PGE₂ from both exogenous and endogenous substrate was suppressed by treatment of the monocytes with indomethacin (10⁻⁶ M).     

Quantitation of luekotrienes C4, D4 amd E4 released by pathophysiological stimuli

In order to examine the modulation of leukotriene (LT) release, the PAF-acether-mediated stimulation of these compounds in rat lung was studied. Release of LTC₄, LTD₄ and LTE₄ in both perfused and chopped lung preparations was measured using HPLC and radioimmunoassay. Pre-incubation or pre-infusion of the tissue with indomethacin and PGE₂ was conducted to investigate the effect of cyclooxygenase inhibitors and products on the lipoxygenase pathway. In addition, the effects of LT levels of pre-incubation with vasoactive intenstinal polypeptide (VIP) in chopped lung were observed.In perfused rat lung, indomethacin reduced the levels of LTC₄ relative to LTD₄ as measured in the first 2 min after stimulation of the lung by PAF-acether. Chopped lung preparations, incubated for 15 min. exhibited higher levels of LTC₄ and LTD₄ in indomethacin-treated samples, this increases being effectively reversed by PGE₂.In the VIP pre-incubation experiments clear inhibition of peptido -leukotriene synthesis was observed, with no LTC₄ and only low levels of LTD₄ and LTE₄ observed in VIP-incubated samples. In preliminary experiments using rabbit C5a des arg and PAF-acether on rabbit lung parenchyma strips to stimulaet LT release, disodium cromoglycate pre-incubation was observed to inhibit this release.Inhibition of the 5-lipoxygenase pathway of PGE₂ is supported by these experiments. VIP appears to act as an inhibitor of LTC₄ and LTD₄ biosynthesis or release in this model. Too little is known that peptidergic actions to postulate a mechanism by which a neuroendocrine peptide exerts control of release of arachidonate metabolites; however, VIP is associated with muscarinic stimulation (1) and has been found in mast cells (2).     

Contraction of isolated airway smooth muscle by synthetic leukotrienes C4 and D4

The pharmacology of leukotrienes (LT) C₄ and D₄ in isolated airway smooth muscle was investigated. In rat trachea, neither LTC₄ or D₄ elicited a response. In contrast, LTC₄ was a potent contractile agonist in guinea-pig trachea, bronchus and parenchymal lung strip. Similar effects were obtained with LTD₄ in trachea and parenchyma. In trachea and bronchus, the concentration-response curve to LTC₄ was biphasic: indomethacin converted the biphasic response curve to a simple sigmoidal shape and enhanced the maximum contractile response. The SRS-A antagonist FPL 55712 antagonized the effect of LTD₄ in both trachea and parenchyma. As regards LTC₄-induced contraction of trachea and bronchus, FPL 55712, depending on concentration, either antagonized, or antagonized and enhanced the maximum contractile response. The enhancement of the maximum contractile response by FPL 55712 was not apparent when indomethacin was present. FPL 55712 failed to antagonize the effect of LTC₄ in parenchyma.     

Bronchoconstrictor effects of leukotriene B4 in the guinea pig

Administration of leukotriene B₄ (LTB₄) to anesthetized spontaneously breathing guinea pigs either by the intravenous or aerosol route produced pronounced changes in pulmonary resistance and dynamic compliance. The effects were short lived and were completely abolished by pretreatment of animals with the cyclooxygenase inhibitor indomethacin. Histological examination of lungs following aerosol administration of LTB₄ showed a pronounced neutrophil infiltration. These results confirm previous studies in which LTB₄ was shown to produce contractions on guinea pig parenchymal strips indirectly by releasing thromboxane A₂.     

Leukotriene and prostaglandin production in rat brain synaptosomes treated with phospholipase A2 neurotoxins and enzymes

β-Bungarotoxin (β-BuTX) and notexin cause an irreversible blockade of neurotransmitter release through specific and potent effects at the presynaptic nerve terminal, however, the mechanism of action in uncertain. We examined the effects of β-BuTX and notexin on LT and PG production in rat cerebrocortical synaptosomes in order to determine if eicosanoid production might mediate or regulate the pharmacological actions of these phospholipase A₂(PLA₂) neurotoxins. The effects of the PLA₂ enzymes isolated from Naja naja atra and Naja nigricollis snake venoms (which are not presynaptic selective) on LT and PG production were compared with the effects of β-BuTX and notexin. N. n. atra PLA₂, β-BuTX, and notexin (all 50 nM) produced a time dependent rise in free fatty acids as measured in synaptic plasma membranes isolated from treated synaptosomes. Both the PLA₂ neurotoxins and enzymes stimulated LTC₄, LTB₄, and PGE₂ production, as measured by radioimmunoassay. In all cases, the PLA₂ enzymes were more potent than the PLA₂ neurotoxins. This observations correlates with their relative enzymatic potencies, as measured by free fatty acid generation. EDTA and BSA antagonized PLA₂ induced LTB₄ production and BSA also antagonized PLA₂ induced PGE₂ production. These results suggest that stimulation of eicosanoid production does not mediate the potent and specific presynaptic actions of β-BuTX and notexin.     

Characterization of leukotriene A4 and B4 bioysnthesis

We have studied LTA₄ and LTB₄ synthesis in a cell-free system from RBL-1 cells. All the enzymes leading to the formation of LTB₄ from arachidonic acid are localized in the soluble fraction (100, 000 x g supernatant) of these cells. The formation of LTA₄ and LTB₄ is complete by 10 min. When we varied the arachidonic acid concentration from 1 to 300 μM, the synthesis of LTB₄ leveled off at 30 μM and of LTA₄ at 100 μM while 5-HETE had not reached a plateau at 300 μM. This enzyme system has the capacity to generate relatively large amounts of 5-HETE and LTA₄ and only a relatively small amount of LTB₄. Therefore, the rate limiting step is not the 5-lipoxygenase, the first step in the pathway, but the conversion of LTA₄ to LTB₄. This is in contrast to cyclooxygenase pathway where the first step is rate limiting. A second addition of arachidonic acid at submaximal concentration for LTA₄ synthesis did not produce any additional LTA₄ or LTB₄. Further study of this phenomenon showed that the 5-lipoxygenase and LTA-synthase were inactivated with time by preincubation with arachidonic acid and that peroxy fatty acids seem to be the inactivating species.