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.     

Positive interaction between leukotriene C4 and histamine and other mediators on vascular tissues

The interaction of leukotriene C₄ (LTC₄) with the contractile activity of histamine (H), serotonin (5HT) and norepinephrine (NE) has been investigated in isolated vascular preparations. Threshold concentration of LTC₄ (5 × 10⁻⁹ M) significantly potentiated the vasoconstricting effect of these compounds on guinea-pig pulmonary artery (GPPA). This phenomenon was long-lasting for H since it was still present 40 min after LTC₄ had been washed. FPL-55712 (10⁻⁵M) counteracted the increased H response on GPPA induced by LTC₄. Potentiation of H activity due to LTC₄ was also observed on guinea-pig thoracic aorta (GPTA) indicating that LTC₄-induced hyperreactivity is not a phenomenon restricted to the pulmonary vascular bed. In the experiments carried out in presence of indomethacin (3 × 10⁻⁶M), LTC₄ still potentiated H-induced vasoconstriction on GPPA, however the time course of the phenomenon was significantly shorter than that observed in absence of the cyclooxygenase inhibitor. The contractile activity of H and NE on guinea-pig portal vein (GPPV) was not potentiated by LTC₄ These results demonstrate that LTC₄ induces hyperreactivity of the arterial vascular tissue to vasoactive compounds and suggest that cysteinyl-leukotrienes may have pathological significance in the hemodynamic changes occurring during anaphylactic reactions. Preliminary experiments carried out on human intralobar pulmonary artery strongly support this hypothesis.     

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.     

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.     

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.     

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.     

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.     

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.     

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% ntact) 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 γ-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.     

The effect of leukotrienes C4 and D4 on the microvasculature of guinea-pig skin

The effects of chemically-synthesised leukotrienes C₄ and D₄ (5(S) hydroxy-6(R)-δ-glutamylcysteinylglycinyl-7,9,11,14-eicosa-⁴tetraenoic acid, LTC₄; 5(S) hydroxy-6(R)-cysteinylglycinyl-7,9,11,14-eicosatetraenoic acid, LTD₄) on the microvasculature have been measured in guinea-pig skin using [¹²⁵I]-albumin accumulation to measure plasma exudation and ¹³³Xe clearance to measure blood flow changes. As previously shown using biosynthetic material, LTD₄ caused vasoconstriction resulting in reduced blood flow. Similarly, LTC₄ was found to have vasoconstrictor activity but was more potent and had a steeper dose-response curve than LTD₄. There was no evidence of conversion of exogenous arachidonic acid to vaso-constrictor activity in the skin $\text{in vivo}$ (in the absence of another stimulus): intradermally injected arachidonic acid produced vasodilatation, but induced little change in blood flow in animals pretreated with indomethacin. The vasodilator effect of arachidonic acid is presumed to be due to conversion to either PGE₂ or PGI₂. These results suggest that cyclo-oxygenase is normally active in the skin, whilst lipoxygenase requires activation in some way. As reported in a previous study, LTD₄ induced plasma exudation when injected into the skin, but pronounced responses could only be induced by LTD₄ mixed with a vasodilator prostaglandin such as PGE₂. In contrast, LTC₄ induced no exudation when tested alone and little when PGE₂ was added. However, evidence was obtained that LTC₄ has some permeability-increasing activity which is marked by its potent vasoconstrictor activity.     

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.     

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.     

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.     

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。     

Synthesis and biological activities of leukotriene F4 and leukotriene F4 sulfone

Leukotriene F₄ (LTF₄ and LTF₄ sulfone have been synthesized and their biological activities determined in the guinea pig. LFT₄ displayed comparable activity to LTD₄ on guinea pig trachea and parenchyma but was less active on the ileum. When injected intravenously into the guinea pig, LTF₄ induced a bronchoconstriction (ED₅₀ 16 μg Kg⁻¹) which was blocked by indomethacin and FPL-55712 and was 50–100 X less potent than LTD₄ in this assay. LTF₄ sulfone was approximately 2–5 times less active than LTF₄ and . When injected into guinea pig skin with PGE₂ (100 ng); LTF₄ and LTF₄ sulfone (10–1000 ng) induced changes in vascular permeability. The order of potency in this assay was LTE₄ sulfone = LTD₄ = LTD₄ sulfone > LTE₄ > LTF₄ = LTF₄ sulfone.     

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.     

Surface lipid composition of C3 and C4 plants

The characteristic surface lipid compositions of several C₃ and C₄ plants are discussed. C₄ plants produce surface lipids (epicuticular waxes) made up of the ubiquitous classes of aliphatic compounds: free fatty acids, aldehydes, primary alcohols, alkanes and aliphatic linear esters. C₃ plants synthesize surface lipids comprising the ubiquitous classes and either of the two following groups of compound: (i) lβ-diketones, hydroxy lβ-diketones, alkan-2-ol esters; (il) ketones and secondary alcohols with the functional group in the middle of the hydrocarbon chain. These features are suggested to represent physioIogical characteristics of the plant and to be related to ecological adaptations. Wax class compositions might also be an ancillary method for defining the C₃ or C₄ mechanism of CO₂ assimilation in cases where uncertainty exists.