Bioconversion of leukotriene C4 by rat glomeruli and papilla

Since leukotriene C4 (LTC4) may be locally synthesized by bone marrow-derived cells infiltrating the kidney in inflammatory renal diseases we examined the in vitro metabolism of exogenously added [3H] LTC4 by rat glomeruli and papilla using radiometric HPLC. Homogenized as well as intact glomeruli converted [3H] LTC4 mainly into [3H] LTE4 (83%) and, at a smaller extent, into [3H] LTD4 (4%). Intact [3H] LTC4 represented 13% of the sum of radioactive leukotrienes. Addition of L-cysteine resulted in accumulation of LTD4. In contrast, there was nearly no conversion of [3H] LTC4 (87% intact) in the presence of homogenized papilla. The metabolism of [3H] LTC4 by the glomeruli was time- and temperature-dependent. The 10,000 g supernatant and pellet of homogenized glomeruli both retained the ability to metabolize [3H] LTC4. The papillary 10,000 g supernatant was inactive, as found for the total homogenate, whereas the papillary 10,000 g pellet separated from its supernatant could transform [3H] LTC4 into its metabolites, LTD4 and LTE4. Addition of increasing amounts of papillary 10,000 g supernatant to homogenized glomeruli progressively protected [3H] LTC4 from its bioconversion. These results demonstrate that both glomeruli and papilla possess the gamma-glutamyl transpeptidase and dipeptidase necessary to process LTC4. However, the enzyme activity of the papilla is unmasked only when the inhibitor present in the 10,000 g supernatant is separated from the enzyme present in the pellet.     

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.     

Leukotriene A4: Enzymatic conversion to leukotriene C4

An unstable epoxide, leukotriene A₄ (5(S)-$\text{trans}$-5,6-oxido-7,9-$\text{trans}$-11,14-$\text{cis}$-eicosatetraenoic acid), was earlier proposed to be an intermediate in the conversion of arachidonic acid into the slow reacting substance (SRS), leukotriene C₄. In the present work synthetic leukotriene A₄ was incubated with human leukocytes or murine mastocytoma cells. A lipoxygenase inhibitor, BW755C, was added in order to prevent leukotriene formation from endogenous substrate. Leukotriene C₄ and 11-$\text{trans}$-leukotriene C₄ were the main products with SRS activity. It was not established whether the 11-$\text{trans}$-compound was formed by isomerization at the leukotriene A₄ or C₄ stage.     

Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies

Background

Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC₄ (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC).

Methods

We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC₄ and LTD₄ by competing their binding to their receptors.

Results

mAbLTC and scFvLTC inhibited their binding of LTC₄ or LTD₄ to CysLT₁ receptor (CysLT₁R) and CysLT₂ receptor (CysLT₂R) overexpressed in Chinese hamster ovary cells. The induction by LTD₄ of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT₁R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC₄- and LTD₄-induced aggregation of mouse platelets expressing CysLT₂R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT₂R antagonists but not to CysLT₁R antagonists.

Conclusions

These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT₁R and CysLT₂R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors.

General significance

mAbLTC can be used in the treatment of inflammatory diseases such as asthma.     

Ethanol stimulates formation of leukotriene C4 in rat gastric mucosa

Ethanol-induced gastric mucosal damage is characterized by microcirculatory changes such as statis and plasma leakage. Sluggish blood flow and statis have also been observed after administration of exogenous leukotriene (LT) C₄. The effect of ethanol on the release of LTC₄ from rat gastric mucosa was therefore investigated. It was found that intragastric instillation of ethanol increases gastric mucosal release of LTC₄ in a dose- and time-dependent manner parallel to the production of gastric lesions. The lipoxugenase inhibitor nordihydroguaiaretic acid (NDGA) and the anti-ulcer drug carbenoxolone (CX) inhibited mucosal release of LTC₄ and simultaneously protected against gastric damage caused by ethanol. It is concluded that increased formation of LTC₄ and /or other 5-lipoxygenase-derived products of arachidonate metabolism may be involved in ethanol-induced gastric damage. Furthermore, inhibition of the 5-lipoxygenase pathway may be an important mechanism of action of gastric protective drugs.     

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.     

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.     

Isolation of leukotriene C4 from human seminal fluid

LTC₄ was isolated and characterized from seminal fluid of seven human volunteers. A compound with a similar retention time of that of synthetic LTC₄ was obtained using reverse-phase high performance liquid chromatography. The ultraviolet absorbance of the extracted substance was identical to synthetic LTC₄. Furthermore this compound contracted the guinea pig ileum and lung parenchymal strip. Its effects were antagonized by the leukotriene antagonist FPL55712. It was concluded that LTC₄ is present in human seminal fluid in very small amounts (about 100 ng/ejaculate). The possible physiological functions of LTC₄ in the reproductive tract area discussed.     

Release of leukotriene C4 from guinea pig trachea

Immunological (ovalbumin) and non-immunological (calcium ionophore A23187) stimulation of guinea pig trachea induces a prolonged contraction that is enhanced by indomethacin (8.5 μM) and inhibited by nordihydroguaiaretic acid (50 μM) pretreatment of the tissue. The mediator released by the above stimuli was identified as leukotriene C₄ by reverse-phase high performance liquid chromatography, and quantitated by bioassay. Indomethacin, and/or arachidonic acid (32.8 μM) did not enhance the release, whereas nordihydrolguaiaretic acid reduced the contraction and release of LTC₄. The results demonstrate the hitherto unproved capability of the large airways to synthesize leukotrienes and emphasize the importance of examining their role in asthma.     

Autoradiographic localization of leukotriene C4 and D4 binding sites in guinea-pig lung

Binding of [3H] leukotriene C4 and D4 to guinea-pig lung sections was charaterised and binding sites were localized by autoradiography. Both leukotrienes bound to guinea-pig lung sections and membranes with high affinity and with similar charateristics to binding in a membrane preparation. Autoradiography revealed that the distribution of LTC4 and D4 binding sites was markedly different. Smooth muscle and epithelium of central and peripheral airways were densely labelled with [3H]LTC4; vascular smooth muscle and alveolar walls were also labelled. With [3H]LTD4, however, there was no detectable labelling of airways or vessels but subtantial labelling of alveolar walls. This lends futher support that LTC4 and LTD4 binding sites differ and may not be identical with functional receptors.     

Interaction between leukotriene C4 and histamine in the guinea-pig

Lipoxygenase metabolites have proposed as potential chemical mediators of the bronchial hyperractivity which characterizes asthma (2,6). In addition to the possibility that leukotrienes (LTs) sensitize airways smooth muscle to the contractile actions of other mediators such as histamine (1–3), a number of studies have provided evidence for LT-induced enhancement of bronchoconstriction by a vagal dependent mechanism (4–6). In the present study the effects of exposure of the airway to LTC₄ on subsequent responsiveness to histamine have been investigated in both and experiments. LTC₄, in a concentration eliciting threshold contractile responses of the isolated trachea (1.7 nM), had no effect on either the EC₅₀ or maximal contractile response to histamine. At a concentration eliciting an approximately EC₅₀ contractile response, LTC₄ (10 nM) shifted the histamine concentration-response curve rightwards altering the maximum response. In anaesthetized, mechanically ventilated guinea pigs LTC₄ (0.1–0.4 nMole/kg, i.v.) injected 20 s beforehand, failed to alter histamine (9–36 nMole/kg, i.v.)-induced bronchoconstriction whereas, under the same conditions, LTD₄ (0.05–0.2 nMole/kg, i.v.) dose-dependently enhanced histamine-induced bronchoconstriction. On the other hand, LTC₄ or LTD₄ (16 uM, 30 s) aerosols potentiated histamine (9.36 nMole/kg, i.v.) in a concentration-dependent manner (Table). Both LTC₄ and LTD₄ aerosols enahance airway reactivity to histamine whereas only LTD₄ has this action when administered intravenously. Neither LTC₄ nor LTD₄ (6) enhances the contractile effects of histamine on isolated airways smooth muscle. It is concluded that the broncho-constriction enhancing action of these leukotrienes may be indirectly mediated.     

Defective inhibition by dexamethasone of leukotriene B4 and C4 production by leukocytes in patients with cystic fibrosis

To investigate the effects of glucocorticoids on leukotriene (LT) generation in patients with cystic fibrosis (CF), we evaluated calcium ionophore A23187-induced LTB₄ and LTC₄ production by leukocytes with and without pretreatment with dexamethasone. The CF patients were in good condition and did not have acute infection. There were no significant differences in LTB₄ and LTC₄ production without dexamethasone pretreatment between the CF patients and controls. However, the ratios of LTB₄ and LTC₄ production by leukocytes preincubated with dexamethasone to those of leukocytes without dexamethasone pretreatment were significantly higher in the CF patients than in the controls (both p < 0.05). Our data suggest that the response of LTB₄ and LTC₄ production to dexamethasone is disturbed in patients with CF. The generation of LTs may be enhanced due to a disturbance in glucocorticoid suppression.     

Involvement of leukotriene C4 in PAF-induced death in mice

The role of leukotrienes (LTs) in the pathogenesis of platelet-activating factor (PAF)-induced death in mice was reinvestigated, since previously reported results are in conflict. A novel 5-lipoxygenase inhibitor, E6080, and a leukotriene antagonist, LY17883, protected mice from PAF-induced death in a dose-dependent manner, while the well-known 5-lipoxygenase inhibitor, AA861, was less effective than E6080. After the intravenous injection of PAF in mice, immunoreactive leukotriene C4 (i-LTC4), which was co-eluted with authentic LTC4 in HPLC, was significantly increased in bronchoalveolar lavage fluid (BALF). Oral administration of E6080 suppressed the increase in i-LTCM4. The results suggest that LTs may play an important role in PAF-induced lethality in mice.     

Leukotriene B3, leukotriene B4 and leukotriene B5; Binding to leukotriene B4 receptors on rat and human leukocyte membranes

Specific high-affinity binding sites for [³H]-leukotriene B₄ have been identified on membrane preparations from rat and human leukocytes. The rat and human leukocyte membrane preparations show linearity of binding with increasing protein concentration, saturable binding and rapid dissociation of binding by excess unlabelled leukotriene B₄. Dissociation constants of 0.5 to 2.5 nM and maximum binding of 5000 fmoles/mg protein were obtained for [³H] leukotriene B₄ binding to these preparations. Displacement of [³H]-leukotriene B₄ by leukotriene B₄ was compared with displacement by leukotriene B₃ and leukotriene B₅ which differ from leukotriene B₄ only by the absence of a double bond at carbon 14 or the presence of an additional double bond at carbon 17, respectively. Leukotriene B₃ was shown to be equipotent to leukotriene B₄ in ability to displace [³H]-leukotriene B₄ from both rat and human leukocyte membranes while leukotriene B₅ was 20–50 fold less potent. The relative potencies for the displacement of [³]-leukotriene B₄ by leukotrienes B₃, B₄ and B₅ on rat and human leukocyte membranes were shown to correlate well with their potencies for the induction of the aggregation of rat leukocytes and the chemokinesis of human leukocytes.     

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.     

Comparison of biological-derived and synthetic leukotriene C4 by fast atom bombardment mass spectrometry

The development of the technique of fast atom bombardment mass spectrometry (FAB-MS) has greatly expanded the capability to analyze non-volatile, complex biochemicals. The structure of leukotriene C₄, a slow reacting substance of anaphylaxis, has recently been postulated as 6-glutathionyl-5-hydroxy-7,9,11,14-eicosatetraenoic acid. Even though LTC₄ has been synthesized, it has not been possible to obtain direct mass spectrometric confirmation of this structure. FAB-MS has indicated that the biological LTC₄ had a molecular weight of 625, identical to that of synthetic LTC₄. The abundance of cationized species with one and two sodium atoms was the major difference between these leukotrienes which is probably a result of the difference in salt content of the purified molecules.     

Prostaglandin E2 and leukotriene C4 levels following different reperfusion periods in rat brain correlated with morphological changes

Prostaglandin E₂ (PGE₂) and leukotriene C₄ (LTC₄) are the metabolites of arachidonic acid (AA) that increase in forebrain following global ischemia and reperfusion. These mediators are highly potent vasoconstrictors of cerebral arteries leading to enhanced vascular permeability that induces the formation of vasogenic edema. In this study, after developing and experimental animal model simulating the concept of ischemic penumbra in the rat, the levels of PGE₂ and LTC₄ produced in the forebrain were measured and the effects of these mediators in short duration and prolonged reperfusion were investigated and then correlated with nueropathological findings. We found statistically significant reduction both in PGE₂ and LTC₄-like activities after just 10 min ischemia (p<0.05, p<0.05). PGE₂-like activity significantly increased in the 4th and 60th min of reperfusion (p<0.05, p<0.05). In the 15th min of reperfusion, PGE₂ was found to be significantly reduced (p<0.005) that may be due to the formation of free oxygen radicals by activation of PG hydroperoxidase reaction that inhibits PGE₂ production in the cylooxygenase pathway. LTs were not significantly increased in any reperfused group. Inhibition of the lipoxygenase pathway of AA metabolism may occur as a result of 15-HPETE (15-hydroperoxyeicosatetraenoic acid) production. Pathologically, edema and degeneration of brain tissue were seen beginning from the 4th min of reperfusion that reached a peak in the 60th min of reperfusion which is in accordance with biochemical changes in the damaged tissue. It is concluded that by preventing the formation of AA metabolites in the early hours of ischemia and reperfusion, it could be possible to increase blood flow in the ischemic penumbra that should limit the infarct area.     

C3与C4植物的环境调控

环境条件决定着不同光合类型植物的地理分布范围和区域 ,一般来说 ,C4 植物分布于高温、强光的环境而 C3植物分布于阴凉、湿润的环境 ,且 C4 比 C3植物光合速率高。但环境条件影响着不同光合类型植物的光合潜能的发挥 ,C4 植物在高温、强光、干旱条件下所表现出来的优势在其它环境条件下未必就显现出来。环境条件甚至可以引起 C3、C4 光合途径间的相互转化 ,这使得目前几种鉴别植物光合类型的方法出现不一致的结果。因此 ,在判断植物的光合类型时 ,要注意多种手段的综合利用 ,同时注意植物所处环境条件的影响。     

Secretogogue responses of leukotriene C4, D4: Comparison of potency in canine trachea

By the use of close arterial injection of leukotrienes into the circulation supplying the upper cervical canine trachea, it has been possible to assess the secretogogue effects of leukotriene C₄, and D₄ on mucus secretion. Both LTC₄ and LTD₄ increased mucus secretion over baseline levels by a statistically significant level (p = < 0.05). LTD₄ was more potent than C₄ with relative potencies of 2500, 320, 630, and 500 based on hillock formation (a measure of secretion) at 1, 2, 3, and 4 minutes after injection. The overall difference in potency in this animal model of mucus production was LTD₄ > C₄ by 1000-fold.