Examination of the effects of piriprost (U-60,257B) on alkaline phosphatase activity of rat endometrial stromal cells in vitro.

Previously, piriprost (U-60,257B; an inhibitor of leukotriene (LT) synthesis) was shown to increase alkaline phosphatase (ALP) activity in cultured endometrial stromal cells (1). The present study investigated the mechanism of action of piriprost in this system. Sensitized rat endometrial stromal cells were isolated and cultured for up to 72 hr with various treatments. Piriprost (100 microM) was found to decrease 5-hydroxyeicosatetraenoic acid (a 5-lipoxygenase product) by 53% after 72 hr which provided evidence that 5-lipoxygenase was being inhibited by piriprost. Lactate dehydrogenase (LDH) activity confirmed that piriprost was not toxic to the cells. The possibility of piriprost acting in an analogous manner with that of PGs was examined. Three microM PGE2 or 20 microM carba-prostacyclin (CP), an analogue of PGI2, maximally increased (p less than 0.01) ALP activity at 72 hr and the further addition of 100 microM piriprost to PGE2 or CP caused an additional, additive increase in ALP activity. This indicated that the mechanism of action of piriprost was probably different from that of PGE2 or PGI2. The possibility that piriprost was shunting arachidonic acid into PG production was examined. Ten microM indomethacin (an inhibitor of PG synthesis) caused a decrease (p less than 0.01) in ALP activity and a 99% reduction in PGE2 at 72 hr. The effects of the combination of 100 microM piriprost and 10 microM IM were statistically additive, suggesting that the effects of piriprost were not due to an increase in PG production. These studies suggest that the effects piriprost on possible in vitro decidualization may be due to inhibition of 5-lipoxygenase.     

Determination of SRS-A release from guinea-pig lungs by a radioimmunoassay

A sensitive radioimmunoassay for leukotrienes (LTs) has been developed. Rabbits were immunized with a conjugate of LTD4 and bovine serum albumin, prepared by using 1,5-difluoro-2,4-dinitrobenzene as the coupling agent. The assay can detect 0.045 pmol LTD4 at a final plasma dilution of 1:72. 50% displacement of bound 3H-LTD4 was obtained with 0.43 +/- 0.03 pmol LTD4. LTC4, LTE4 and LTF4 cross-react 159%, 57% and 85%, respectively, whereas LTB4, 5-HETE and prostaglandins did not. The assay was validated by measuring the antigen-induced release of LTs from sensitized guinea-pig chopped lungs. High correlation (0.9434, p less than 0.05) was found when LTs were simultaneously determined by this assay and a bioassay on guinea pig ileum.     

Synthesis and metabolism of leukotrienes by human endothelial cells: influence on prostacyclin release

The synthesis and metabolism of leukotrienes (LTs) by endothelial cells was investigated using reverse-phase high-performance liquid chromatography. Cells were incubated with [14C]arachidonic acid. LTA4 or [3H]LTA4 and stimulated with ionophore A23187. The cells did not synthesize leukotrienes from [14C]arachidonic acid. LTA4 and [3H]LTA4 were converted to LTC4, LTD4, LTE4 and 5,12-diHETE. Endothelial cells metabolized [3H]LTC4 to [3H]LTD4 and [3H]LTE4. The metabolism of [3H]LTC4 was inhibited by L-serine-borate complex, phenobarbital and acivicin in a concentration-related manner, with maximal inhibition occurring at a concentration of 0.1 M, 0.01 M and 0.01 M, respectively. LTC4, LTB4 and LTD4 stimulated the synthesis of prostacyclin, measured by radioimmunoassays as 6-keto-PGF1 alpha. The stimulation by LTC4 was greater than that by LTD4 or LTB4. LTE4, 14,15-LTC4 and 14,15-LTD4 failed to stimulate the synthesis of prostacyclin. LTD4 and LTB4 also stimulated the release of PGE2, whereas LTC4 did not. Serine-borate and phenobarbital inhibited LTC4-stimulated synthesis of prostacyclin in a concentration-related manner. They also inhibited the release of prostacyclin by histamine, A23187 and arachidonic acid. Acivicin had no effect on the release of prostacyclin by LTC4, histamine or A23187. Furthermore, FPL-55712, an LT receptor antagonist, inhibited LTC4-stimulated prostacyclin synthesis but had no effect on histamine-stimulated release of prostacyclin or PGE2. Indomethacin inhibited both LTC4- and histamine-stimulated release. The results show that (a) endothelial cells metabolize LTA4, LTC4 and LTD4 but do not synthesize LTs from arachidonic acid; (b) LTC4 act directly at the leukotriene receptor to stimulation prostacyclin synthesis; (c) the presence of the glutathione moiety at the C-6 position of the eicosatetraenoic acid skeleton is necessary for leukotriene stimulation of prostacyclin release; and (d) the metabolism of LTC4 to LTD4 and LTE4 does not appear to alter the ability of LTC4 to stimulate the synthesis of PGI2.     

The combined use of isolated strips of guinea-pig lung parenchyma and ileum as a sensitive and selective bioassay for leukotriene B4

The biological effects of leukotriene (LT)B4 were compared, on a molar basis, with those of LTC4, LTD4, LTE4, 5-hydroxyeicosatetraenoic acid (5-HETE), PGD2, PGE1, PGE2, PGF2 alpha, PGI2, 6-oxo-PGF1 alpha, bradykinin (BK) and angiotensin II (Ang II) on isolated strips of guinea-pig lung parenchyma (GPP) and ileum smooth muscle (GPISM) superfused in series. LTB4 was similar to LTC4 and LTD4 on GPP, in relation to potency and contractions induced, but differed from LTE4 in being ten times more active and causing contractions of a much shorter duration of action on this tissue. However, unlike the other LTs, LTB4 produced contractions which were resistant to FPL 55712 (1.9 microM) and, when given repeatedly, caused tachyphylaxis in GPP. LTB4 was considerably more active on GPP than the other substances investigated. Further, PGD2, PGF2 alpha and PGI2 contracted GPP, the order of potency being PGD2 greater than PGF2 alpha approximately equal to PGI2, whereas PGE1 and PGE2 relaxed this tissue. In contrast to all other agonists tested which contracted GPISM, LTD4 displaying the highest activity, LTB4 was inactive on this tissue. 5-HETE and 6-oxo-PGF1 alpha were inactive on both GPP and GPISM. On the basis of differential effects of LTB4 on GPP and GPISM, this assay represents a simple and selective means to distinguish LTB4-like materials from other naturally-occurring substances likely to be generated in inflammatory fluids.     

Characterization of sulfidopeptide leukotriene responses in sheep tracheal smooth muscle in vitro.

We have investigated the effects of leukotrienes (LTs) on isolated tracheal smooth muscle from sheep sensitive to Ascaris suum antigen. LTC4 and LTD4 produced dose-dependent contractions of sheep trachea, but LTE4 was virtually inactive. YM-17690, a non-analogous LT agonist, produced no contractile response up to 100 microM. Indomethacin (5 microM) had no effect on LTC4- and LTD4-induced contractions. L-Serine borate (45 mM), an inhibitor of gamma-glutamyl transpeptidase, shifted the dose-response curve of LTC4 to the left by 161-fold, and L-cysteine (6 mM), an inhibitor of aminopeptidase, shifted the dose-response curves of LTC4 and LTD4 to the left by 67- and 23-fold, respectively. YM-16638 (1 microM), an LT antagonist, shifted the dose-response curves of LTC4 and LTD4 to the right with pKB values of 6.57 and 7.13, respectively. YM-16638 did not affect LTC4-induced contractions of L-serine borate-treated tissues, indicating that the compound acts only on LTD4 receptors in sheep trachea, LTE4 (1 microM) shifted the dose-response curves of LTC4 and LTD4 to the right with pKB values of 6.87 and 7.31, respectively. YM-17690 (10 microM) showed effects similar to LTE4, suggesting that the compound acts as an LTE4 agonist in sheep trachea. These results suggest that in sheep tracheal smooth muscle (a) LTC4 and LTD4 produce contractions, (b) these LT-induced contractions are not mediated by cyclooxygenase products, (c) LTC4 is converted to LTD4 and then to LTE4, and (d) the potency of the LTC4- and LTD4-induced contractions is increased when their conversion to LTE4 is inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization using selected antagonists of the leukotriene receptors mediating contraction of guinea-pig trachea

The effects of six leukotriene (LT) antagonists on LTC4-, D4- and E4-induced contraction of isolated guinea-pig tracheal spirals were examined. Concentration-response effects of the leukotrienes were determined by cumulative addition in the presence of indomethacin (5 microM) alone for LTE4, or with 10 mM of either glutathione or L-cysteine to inhibit metabolism of LTC4 or LTD4, respectively. Concentration-response curves to the LTs were obtained in the absence and presence of Wy-45,911, Wy-44,329, FPL-55,712, Ly-171,883, Wy-48,252 and ICI-198,615 representing three structurally different chemical groups of LT antagonists. At 30 microM, the antagonists produced little or no antagonism of LTC4-induced contractions. Analysis of the Schild plots for antagonism of LTD4 and E4 suggested two receptors for the agonist effects of LTD4 and a single receptor for the agonist effects of LTE4. Comparison of pA2 values for Wy-45,911, FPL-55,712, LY-171,883 and Wy-48,252 provided evidence that LTE4 is acting at the antagonist high affinity LTD4 receptor to produce contractile effects. From the data, we conclude that there are three LT receptors (one for LTC4 and two LTD4 subtypes) through which exogenously applied LTs evoke contraction of the isolated guinea-pig trachea.     

Characteristics of formation and further metabolism of leukotrienes in the chopped human lung

The bronchoconstrictive leukotrienes (LTs) LTC4, LTD4 and LTE4 (cysteinyl-LTs) and the chemoattractant LTB4 were formed in chopped human lung stimulated by the calcium ionophore A23187, or supplied with the precursor LTA4. In contrast, challenge with anti-IgE exclusively induced release of cysteinyl-LTs, indicating that LTB4 is not released as a primary consequence of IgE-mediated reactions in the human lung. Furthermore, several differences were observed with respect to formation and further conversion of LTB4 and LTC4 in the chopped lung preparation. Thus, exogenous [1-14C]arachidonic acid was dose-dependently converted to radioactive LTB4, whereas the cysteinyl-LTs released were not radiolabeled and the amounts of LTC4, D4 and E4 were not influenced by addition of increasing concentrations of arachidonic acid. LTC4 was rapidly and completely converted into LTD4 and LTE4, with no further catabolism of LTE4 within 90 min. The metabolism of LTB4 was much slower than that of LTC4. Thus, following a 60 min incubation approx. 25% of the material remained as LTB4, whereas 35% was omega-oxidized and 40% eluted on RP-HPLC as two unidentified peaks.     

Leukotriene C4 action and metabolism in the isolated perfused bullfrog heart

The effects of leukotrienes (LTs) have been widely studied in the isolated perfused mammalian heart; however, little is known about the effect or metabolism of LTs in the isolated bullfrog heart. Isolated perfused bullfrog hearts were administered randomized doses of LTC4, LTD4, or LTE4. The cardiac parameters of heart rate, developed tension, and its first derivative (dT/dt) were recorded. LTC4 was the most potent of the leukotrienes tested in eliciting positive inotropic effects. LTD4 and LTE4 were equally effective but about one order of magnitude less potent than LTC4. None of the LTs showed any chronotropic effects in this preparation. A series of [3H]LTC4 metabolism experiments were carried out using whole perfused hearts and minced bullfrog heart tissue. Isolated perfused bullfrog hearts administered [3H]LTC4 converted significant amounts to [3H]LTD4, and to a lesser degree, [3H]LTE4, during the 6-min course of collection. Both minced atrial and ventricular tissue converted [3H]LTC4 to radioactive metabolites that co-migrated with authentic LTD4 and LTE4 standards. In both tissues, the major product was [3H]LTD4, with smaller amounts of [3H]LTE4 produced. The atrium converted significantly more [3H]LTC4 to its metabolites than did the ventricle. The metabolism of [3H]LTC4 to [3H]LTD4 by both tissues was virtually abolished in the presence of serine borate. Cysteine had no effect on [3H]LTE4 production. The data in this study demonstrate that leukotrienes have the opposite inotropic effect on the heart when compared with mammals. Also in contrast to mammals, frogs metabolize LTC4 to a less potent compound and may use the LTC4 to LTD4 conversion as a mechanism of LTC4 inactivation.     

Pharmacology of peptide leukotrienes on ferret isolated airway smooth muscle

The contractile activities of peptide leukotrienes (LT) on isolated spiral strips of ferret trachea were characterized pharmacologically. LTC4 and LTD4 contracted ferret tracheal strips in a concentration-related manner and were 3- to 8-fold more potent than carbachol. In contrast, high concentrations of LTE4 evoked either weak contractions or none at all, whereas LTC4 and D4 were partial agonists compared to carbachol. In tissues which were unresponsive to LTE4, this compound antagonized contractile responses to LTC4 and D4 in an apparently competitive manner: Carbachol-induced contractions were not altered by LTE4. The cyclooxygenase inhibitor, indomethacin (5 microM), LT antagonist, FPL55712 (10 microM), atropine (1 microM), phenoxybenzamine (10 microM), and LTB4 (10 microM) failed to alter LTC4 and D4 concentration-response curves. The results indicate that ferret trachea is sensitive to the contractile activity of LTC4 and LTD4 but not LTE4. The LT-induced contractions appear to be mediated by a direct action of the LT rather than indirectly through release of secondary mediators such as thromboxane, prostaglandin, or acetylcholine. LT receptors in ferret trachea are insensitive to FPL55712 but are antagonized by LTE4.     

Evaluation and interference of serum and skin lesion levels of leukotrienes in patients with eczema

To evaluate the levels of leukotrienes (LTs), interleukin-2 (IL-2), interleukin-4 (IL-4), interferon-gamma (IFN-gamma) in patients with eczema and observe the effects inversed by mizolastine. Serum LTB4, LTC4, IL-2, IL-4, IFN-gamma and urinary LTE4 levels were detected by enzyme-linked immunosorbent assay (ELISA) and LTB4, LTC4, LTE4 concentrations of cutis tissue were measured by reverse-phase high-pressure liquid chromatography (RP-HPLC) in 10 eczema patients and 10 healthy volunteers. Eczema patients received mizolastine 10 mg once a day for 5 days, respectively, for comparison between before and after treatment. The above markers were assayed again after treatment. Serum LTB4, LTC4, IL-2, IFN-gamma and urinary LTE4 and skin tissue LTB4, LTC4, LTE4 levels in patients are higher than those in healthy volunteers significantly (P < 0.05). But serum IL-4 level did not show significant difference between patients and normal controls (P > 0.05). Mizolastine significantly reduced serum LTB4 and IFN-gamma levels as well as skin lesion LTB4, LTC4, LTE4 concentrations. LTs are involved in the pathogenesis of eczema. Mizolastine clearly reduces LTs levels in skin lesion.     

Prolonged pulmonary hypertension caused by platelet-activating factor and leukotriene C4 in the rat lung.

Platelet-activating factor (PAF) and leukotrienes (LTs) are potent pulmonary hypertensive and inflammatory mediators produced by the lung. Previously we showed that a rapid injection of PAF into the pulmonary artery of an isolated rat lung produced an extended elevation in mean pulmonary arterial pressure (PAP). The objective of the present study was to determine whether the extended pressor response induced by PAF was caused by prolonged activation of the 5-lipoxygenase pathway or slow clearance of LTs from the lung parenchyma. Rat lungs were perfused with a nonrecirculating physiological salt solution that contained indomethacin and albumin. Five minutes after a rapid injection of PAF into the pulmonary artery catheter, the following elevations (mean % above baseline) were observed: PAP (83%), LTB4 (3,260%), LTC4 (1,490%), LTD4 (970%), and LTE4 (1,500%). At 20 min these levels declined but were still significantly elevated above baseline. The 5-lipoxygenase inhibitor diethylcarbamazine (DEC), administered before the PAF injection, inhibited the elevations of PAP and all LTs. DEC administration that began 5 min after PAF reduced PAP and only LTC4 levels at 20 min in comparison to lungs with no DEC. The 5-lipoxygenase-activating protein inhibitor MK886, administered orally 2-6 h before perfusion, also inhibited the pressor response to PAF as well as LT production, as did DEC. We conclude that 1) the extended pulmonary hypertension induced by PAF was caused mainly by prolonged activation of 5-lipoxygenase with LTC4 production, 2) the relative overall lung clearance of LTB4, LTD4, and LTE4 was slower than that of LTC4, and 3) LTB4, LTD4, and LTE4 had no appreciable pressor effect.     

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 in vitro. 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.     

Influences of salts on high-performance liquid chromatography of leukotrienes

The effects of concentrations and kinds of salts on the resolution of leukotrienes (LT) C4, D4, E4, and B4 were investigated by two kinds of reversed-phase high-performance liquid chromatography columns (muBondapak C18 and Novapak C18). When a mobile phase (acetonitrile/methanol/water) with a lower concentration of acetic acid (0.02-0.1%) at pH 5.6 was used, LTC4 and LTD4 were not eluted from the muBondapak column. On the Novapak column, LTC4 and LTD4 were eluted, but they were poorly resolved. When the concentration of acetic acid in the mobile phase was raised to 1.0% and adjusted to pH 5.6 with ammonium hydroxide or triethylamine, excellent resolution of LTs was obtained. Sodium hydroxide was, to some extent, useful for the pH adjustment of the mobile phase. Sodium chloride could not be substituted for acetic acid-ammonium hydroxide or -triethylamine salt. The resolution of LTC4, LTD4, and LTE4 was affected more strongly than that of LTB4 by changes of concentrations and kinds of salts. When the acetonitrile/methanol/water/acetic acid solvent system adjusted to pH 5.6 with triethylamine was applied to the analysis of the leukotrienes produced from rat peritoneal cells with stimulation of calcium ionophore A23187, de novo-synthesized LTC4, LTD4, LTB4, and isomers were clearly separated. This solvent system may be useful for the investigation of variations in the synthesis of subclasses of LTs with different stimuli and under different circumstances.     

Carboxypeptidase A-catalyzed direct conversion of leukotriene C4 to leukotriene F4

Leukotrienes (LTs) are 5-lipoxygenase (5-LO)-derived arachidonic metabolites that constitute a potent set of lipid mediators produced by inflammatory cells. Leukotriene A(4), a labile allylic epoxide formed from arachidonic acid by dual 5-LO activity, is the precursor for LTB(4) and LTC(4) synthesis. LTC(4) is further transformed enzymatically by the sequential action of gamma-glutamyltranspeptidase and dipeptidase to LTD(4) and LTE(4), respectively. In this report, we present evidence that bovine pancreatic carboxypeptidase A (CPA), which shares significant sequence homology with CPA in mast cell granules, catalyzes the conversion of LTC(4) to LTF(4) via the hydrolysis of an amide bond. The identity of CPA-catalyzed LTC(4) hydrolysis product as LTF(4) was confirmed by several analytical criteria, including enzymatic conversion to conjugated tetraene by soybean LO, conversion to LTE(4) by gamma-glutamyltranspeptidase, cochromatography with the standard LTF(4) and positive-ion fast-atom bombardment mass spectral analysis. Thus, it appears that the physiological significance of this single-step transformation may point toward a major cellular homeostatic mechanism of metabolizing LTC(4), a potent bronco- and vasoconstrictor, to a less potent form of cysteinyl LTs.     

A simple and sensitive radioreceptor assay for leukotrienes

A simple and sensitive radioreceptor assay (RRA) for leukotrienes (LTs) was developed using a highly specific [3H]leukotriene D4 (LTD4) binding to guinea pig lung membrane homogenates. The assay can detect down to 0.15 pmol of LTD4. The values for fifty percent inhibition of bound [3H]LTD4 was 1.5 nM for LTD4, 45 nM for LTC4 and 24 nM for LTE4. LTB4 at 3.0 X 10(-5)M had no effect on [3H]LTD4 binding. The RRA for LTs in the absence of serine-borate complex was bi-specific for both LTC4 and LTD4. However, in the presence of 20 mM serine-borate this method was highly specific for LTD4. Recovery rate averaged 87.2% after ethanol extraction and evaporation of known amounts of LTD4. When the radioreceptor assay and radioimmunoassay data for leukotriene levels in the samples were compared to each other, an excellent correlation was observed with a correlation coefficient 'r' of 0.992. The assay was also validated by quantitation of Lts released from human granulocytes stimulated with calcium ionophore, A23187. The method is simpler, less expensive, and more specific for LTD4 than the other methods such as high pressure liquid chromatography and radioimmunoassay and is suitable for routine measurement of either LTD4 specifically or LTC4 plus LTD4 simultaneously in one cell system.     

Synthesis and metabolism of leukotrienes in gamma-glutamyl transpeptidase deficiency

Leukotrienes (LTs) are active lipid mediators derived in the 5-lipoxygenase pathway. LTC(4), the primary cysteinyl LT, is cleaved by gamma-glutamyl transpeptidase (GGT), resulting in LTD(4). We studied the synthesis and metabolism of LTs in three patients with GGT deficiency. LTs were analyzed in urine, plasma, and monocytes after HPLC separation by enzyme immunoassays, radioactivity detection, and electrospray tandem mass spectrometry. Analysis of LTs in urine revealed increased concentrations of LTC(4) (12.8-17.9 nmol/mol creatinine; controls, <0.005 nmol/mol creatinine), whereas LTE(4) was below the detection limit (<0.005 nmol/mol creatinine; controls, 32.2 +/- 8.6 nmol/mol creatinine). In plasma of one patient, LTC(4) was found to be increased (17.3 ng/ml; controls, 9.6 +/- 0.4 ng/ml), whereas LTD(4) and LTE(4) were below the detection limit (<0.005 ng/ml). LTB(4) was found within normal ranges. In contrast to controls, the synthesis of LTD(4) and LTE(4) in stimulated monocytes was below the detection limit (<0.1 ng/10(6) cells; controls, 37.1 +/- 4.8 cells and 39.4 +/- 5.6 ng/10(6) cells, respectively). The formation of [(3)H]LTD(4) from [(3)H]LTC(4) in monocytes was completely deficient (<0.1%; controls, 85 +/- 7%). Our data demonstrate a complete deficiency of LTD(4) biosynthesis in patients with a genetic deficiency of GGT. GGT deficiency represents a new inborn error of cysteinyl LT synthesis and provides a unique model in which to study the pathobiological coherence of LT and glutathione metabolism.     

The biotransformation in vitro of cysteinyl leukotrienes in blood of normal and asthmatic subjects

The metabolism of exogenous leukotriene C4 (LTC4), LTD4 and LTE4 (10(-8) M) was studied in vitro in blood of normal and asthmatic subjects for up to 2 hr by reverse-phase high performance liquid chromatography. In whole blood, incubation of LTC4 (T1/2 = 11.5 min) resulted in the formation of LTD4 and LTE4 whose biosynthesis was inhibited by serine borate (30 mM). Similar experiments performed with LTD4 (T1/2 = 5 min) produced a single metabolite (LTE4) which was inhibited by L-cysteine (10 mM). On the other hand, LTE4 represented a highly stable product in our in vitro system. The bioconversion of LTC4 or LTD4 was slower in plasma but this effect appeared more pronounced for the cysteinylglycinyl derivative. The bioconversion of LTD4 in whole blood or plasma was almost twice as rapid as LTC4. Experiments performed with asthmatic blood showed no significant difference in the survival of LTC4. These results suggest that blood may play a role in regulating the bioavailability of cysteinyl-containing LTs which could be of relevance to their excretion in man.     

Leukotrienes cause eosinophil emigration into conjunctival tissue

The ability of LTB4, LTC4, the 5S,6R and 5R,6S LTD4 stereoisomers, and LTE4 to evoke leukocyte infiltration into the conjunctiva was demonstrated in the guinea pig by histological and light microscopy techniques. LTD4 and LTE4 demonstrated a dose-dependent and predominantly eosinophilic infiltrate over the selected dose range (10 ng to 1000 ng), while there was only a minimal response to LTC4. LTB4 produced marked eosinophil infiltrates only at the highest dose; scattered neutrophil infiltrates were also noted at the high dose of LTB4. The 5R,6S LTD4 stereoisomer did not evoke any leukocyte infiltration. The SRS-A antagonist, FPL 55712, abolished peptidoleukotriene-induced eosinophil emigration, and indomethacin pre-treatment had no inhibitory effect, indicating direct mediation of this response by LTs. Histamine caused a comparable eosinophilia over a dose range of 10 micrograms to 1000 micrograms. LT-induced eosinophil emigration was directed to the conjunctival epithelium; the cells appeared intact and no tissue damage was observed. These results may have relevance in the areas of allergic conjunctivitis and asthma research.     

Leukotriene receptors: classification,gene expression,and signal transduction

Leukotrienes (LTs) are potent pro-inflammatory mediators derived from arachidonic acid by the action of 5-lipoxygenase. There are two groups of LTs: LTB(4) and cysteinyl LTs (LTC(4), LTD(4), and LTE(4)). Both of them play important roles in many inflammatory diseases and allergic responses. Recently, their G-protein coupled receptors have been cloned. The identification of these receptors enables us to analyze their gene structures, regulation of expression, and signal transduction in the cells, and it also leads to the development of useful antagonists. Some LT receptors have been disrupted by gene targeting. Such studies may reveal novel functions of leukotrienes, confirming deeper viewpoints for further research.     

Relationship between leukotriene A4 metabolism and biological activity

The data on the pharmacology of leukotrienes showed that LTA4, LTC4 and LTD4 were equipotent on the guinea-pig lung parenchyma whereas LTB4 was slightly less active. However, on the trachea, the myotropic activity of LTC4 and LTD4 was equivalent and higher than LTB4 and LTA4. The potency of these compounds was also different on the ileum where LTD4 was more active than LTC4; at the concentration used, LTA4 and LTB4 were inactive on this tissue. These results suggested that the transformation of leukotrienes by the smooth muscle preparations was a prerequisite for its biological activity. To verify this hypothesis, LTA4 (100 ng) was incubated for 10 min. with 20,000 g supernatants of homogenates of guinea-pig lung parenchyma, trachea and ileum; the metabolites were analysed by bioassay using strips of guinea-pig ileum and lung parenchyma in a cascade superfusion system and by RP-HPLC. Homogenates of lung parenchyma rapidly transformed LTA4 to LTB4, LTC4, LTD4 and LTE4, which is in agreement with the myotropic potency of these leukotrienes on the lung parenchymal strip. Conversely, incubation of LTA4 with homogenates of guinea-pig ileum showed the formation of LTB4 and its isomers which are inactive on this preparation. Similarly, incubation of homogenates of trachea with LTA4 led to the formation of LTB4; this finding is again in agreement with the potency of these two leukotrienes on the trachea. Our results suggest that the myotropic activity and potency of LTA4 is related to the tissue levels of enzymes which catalyse its transformation.