Correlation between the myotropic activity of leukotriene A4 on guinea-pig lung, trachea and ileum and its biotransformation in situ

Leukotrienes A4 and D4 displayed equivalent myotropic activity on guinea pig lung parenchyma strips. However, on the trachea, the activity of LTD4 was much higher than that of LTA4. The potencies of these two leukotrienes were also different on strips of longitudinal muscles of the ileum where LTD4 was very active whereas LTA4 was inactive. Since the activities of both leukotrienes were blocked by FPL-55712, our results suggested that the transformation of LTA4 by the smooth muscle preparations was a prerequisite to its biological activity. LTA4 was then incubated for 10 min with homogenates of guinea pig lung parenchyma, trachea and longitudinal muscles of ileum, and the metabolites were analysed by bioassay using strips of guinea pig ileum and lung parenchyma in a cascade superfusion system and also by reversed phase high performance liquid chromatography (RP-HPLC). Homogenates of lung parenchyma rapidly transformed LTA4 to LTB4, LTC4, LTD4 and LTE4. Incubation of LTA4 with homogenates of trachea or of the longitudinal muscles of ileum showed the formation of LTB4 and its isomers but no significant amount of peptido-leukotrienes were detected. These findings reveal that LTA4 undergoes distinctly different metabolic transformations in these tissues which correspond to the biological activities of the products recovered. These results strongly suggest that the myotropic activity and potency of LTA4 is related to the tissue levels of enzymes which catalyse its biotransformation.     

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.     

Correlation between the myotropic activity of leukotriene A4 on guinea-pig lung, trachea and ileum and its biotransformation in situ

Leukotrienes A₄ and D₄ displayed equivalent myotropic activity on guinea pig lung parenchyma strips. However, on the trachea, the activity of LTD₄ was much higher than that of LTA₄. The potencies of these two leukotrienes were also different on strips of longitudinal muscles of the ileum where LTD₄ was very active whereas LTA₄ was inactive. Since the activities of both leukotrienes were blocked by FPL-55712, our results suggested that the transformation of LTA₄ by the smooth muscle preparations was a prerequisite to its biological activity. LTA₄ was then incubated for 10 min with homogenates of guinea pig lung parenchyma, trachea and longitudinal muscles of ileum, and the metabolites were analysed by bioassay using strips of guinea pig ileum and lung parenchyma in a cascade superfusion system and also by reversed phase high performance liquid chromatography (RP-HPLC). Homogenates of lung parenchyma rapidly transformed LTA₄ to LTB₄, LTC₄, LTD₄ and LTE₄. Incubation of LTA₄ with homogenates of trachea or of the longitudinal muscles of ileum showed the formation of LTB₄ and its isomers but no significant amount of peptido-leukotrienes were detected. These findings reveal that LTA₄ undergoes distinctly different metabolic transformations in these tissues which correspond to the biological activites of the products recovered. These results strongly suggest that the myotropic activity and potency of LTA₄ is related to the tissue levels of enzymes which catalyse its biotransformation.     

The mechanism of action of leukotrienes C4 and D4 in guinea-pig isolated perfused lung and parenchymal strips of guinea pig, rabbit and rat

The biological actions of pure slow-reacting substance of anaphylaxis (SRS-A) from guinea-pig lung, pure slow-reacting substance (SRS) from rat basophilic leukaemia cells (RBL-1) and synthetic leukotrienes C4 (LTC4) and D4 (LTD4) have been investigated on lung tissue from guinea pig, rabbit and rat. In the guinea pig, the leukotrienes released cyclo-oxygenase products from the perfused lung and contracted strips of parenchyma. The effects of SRS-A, SRS and LTD4 were indistinguishable. LTC4 and LTD4 had similar actions although LTD4 was more potent than LTC4. Indomethacin (1 microgram/ml) inhibited the release of cyclo-oxygenase products from perfused guinea-pig lung and caused a marked reduction in contractions of guinea-pig parenchymal strips (GPP) due to LTC4 and LTD4. The residual contraction of the GPP was abolished by FPL 55712 (0.5 - 1.0 microgram/ml). It appears, therefore, that a major part of the constrictor actions of LTC4 and LTD4 in guinea-pig lung are mediated by myotropic cyclo-oxygenase products, i.e. thromboxane A2 (TxA2) and prostaglandins (PGs). In rabbit and rat lung, however, SRS-A, SRS and the leukotrienes were much less potent in contracting parenchymal strips and there was little evidence of the release of cyclo-oxygenase products. FPL 55712 at a concentration of 1 microgram/ml failed to antagonise leukotriene-induced contractions.     

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.     

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.     

Antagonistic effect of KC-404, a new anti-asthmatic agent, on leukotriene D4-induced contractile responses in isolated guinea pig smooth muscles

The inhibitory effects of KC-404, a novel clinically available anti-asthmatic drug, on leukotriene(LT) D4-, LTC4-, histamine- and acetylcholine(ACh)-induced contractile responses in isolated guinea pig lung parenchymal, tracheal and ileal longitudinal strips were compared using an organ bath system. In lung parenchyma, KC-404 antagonized LTD4 in a competitive fashion, whereas it antagonized histamine noncompetitively. The pA2 value against LTD4 was 7.39. KC-404 hardly antagonized LTC4 and ACh. A ranked order of potency estimated from its minimum effective concentrations (MEC) was LTD4 greater than histamine greater than LTC4 greater than ACh. In trachea, KC-404 antagonized LTC4 and LTD4 in a competitive fashion, while it antagonized histamine noncompetitively. The pA2 values against LTC4 and LTD4 were 5.99 and 6.51, respectively. KC-404 hardly antagonized ACh. A ranked order of the potency estimated from MEC was LTD4 greater than LTC4 greater than histamine greater than ACh. The pA2 values of KC-404 against LTD4 in lung parenchyma and trachea were little or not altered, while its inhibitory effect on histamine-induced contraction in trachea was markedly diminished by the pretreatment of tissues with indomethacin. In ileum, KC-404 noncompetitively antagonized all of the agonists used. A ranked order of the potency estimated from pD2 values was LTD4 divided by LTC4 greater than histamine greater than ACh. These results suggest that KC-404 is a selective antagonist of LTD4 and that it might interact with LTD4 receptor in airway smooth muscles but not in ileum. Another possibility that the drug might interact with LTD4 specific excitation-contraction coupling mechanism was also discussed.     

Further studies on the mechanism of action of leukotrienes and histamine on guinea pig lung parenchyma. Role of calcium, phospholipase and methyltransferase

The contractile activity of leukotriene B4 (LTB4), leukotriene D4 (LTD4) and histamine on strips of guinea pig lung parenchyma was shown to be dependent on the calcium concentrations of the Krebs solution. The calcium channel blocker verapamil (2.0 to 15 microM) had an additive effect on the inhibitory activity of low calcium (0.1 mM) on contractions of guinea pig parenchyma to leukotrienes and histamine. Cobalt chloride, a divalent cation, also produced dose-dependent reductions of the myotropic activities of LTB4, LTD4 and histamine. An antagonist of calmodulin, trifluoperazine (1-200 microM), dose-dependently inhibited the contractile activity of the three agonists on the parenchyma strip. The IC50 of this compound for inhibition of histamine was much lower (2-3 microM) than the IC50 for inhibition of leukotrienes (75 microM). Valinomycin, a potassium ionophore, also interfere with the contractile activities of leukotrienes and histamine whereas a blocker of sodium channel, tetrodotoxin, had no effect on the activity of these agonists. Furthermore, an inhibitor of methyltransferase, 3-deazaadenosine, significantly diminished the responses of the parenchyma to leukotrienes and histamine. These results confirmed the important role of extracellular and intracellular calcium in the myotropic activity of leukotrienes and histamine in guinea pig lungs and showed that compounds which interfere either directly or indirectly with calcium mobilization into the lung smooth muscles, decreased the tissue responsiveness.     

Comparative activity of natural mono-, di- and tri-hydroxy derivatives of arachidonic acid on guinea-pig lungs: myotropic effect and stimulation of cyclooxygenase activity

The activity of natural 5,6-Dihydroxy-eicosatetraenoic acid (5,6-DiHETE; 2 isomers), 5S,15S-DiHETE, 8S,15S-DiHETE, 5S,12S-DiHETE, delta 6-trans-leukotriene B4, 12-epi-delta 6-trans-leukotriene B4, omega-hydroxy-leukotriene B4, omega-carboxy-leukotriene B4, 15S-hydroxyeicosatetraenoic acid (15S-HETE), 12S-HETE, 5S-HETE and 12S-hydroxy-heptadecatrienoic acid was compared to LTB4 on the guinea-pig lung parenchymal strip and on the release of prostaglandins and thromboxanes by the perfused guinea-pig lungs. The omega-hydroxy-LTB4 appeared more potent than LTB4 both for inducing a contraction and for releasing prostanoids whereas the omega-carboxy-LTB4 was much less active on the parenchyma and did not release prostanoids at the dose used. All other hydroxy acids tested were either very weakly active or inactive in the two systems used with the exception of the 5,6-DiHETEs which showed significant activity. These di-hydroxy acids induced contractions of the lung parenchymal strip which could be blocked by FPL-55712 but were inactive on the guinea-pig ileum. The 5S-HETE, 12S-HETE and 15S-HETE were also tested for possible myotropic activity on selected smooth muscle preparations. Our results provide further informations on the structural requirements for LTB4 (and other hydroxy acids) actions on the guinea-pig lungs.     

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.     

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.     

Comparative biological activities of the four synthetic (5,6)-dihete isomers

(5,6)-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acids [5,6)-DiHETEs) were synthesized and separated into four pure diastereoisomers. They were tested for comparative binding affinities to leukotriene receptors (LTC4, LTD4, LTB4) in guinea pig lung membranes. Only (5S,6R)-DiHETE was recognized by the LTD4 receptor, the other receptors interacted with neither of the four isomers. (5S,6R)-DiHETE also contracted ileum in vitro and this effect was inhibited by the LTD4 receptor antagonists ICI 198,615 and SKF104,353. These data suggest that the bioproduct (5S,6R)-DiHETE generated by enzymatic conversion of LTA4 could have some LTD4-like activity when produced in large concentrations.     

Specific leukotriene D4 receptors on guinea-pig alveolar macrophages

Specific binding sites for (3H)-leukotriene D4 (LTD4) were identified on guinea-pig alveolar macrophages (GPAMs) using high specific activity (3H)-LTD4, in the presence or absence of unlabelled LTD4. The time required for (3H)-LTD4 binding to reach equilibrium was approximately 15 min at 0 degrees C. The binding was saturable, reversible and specific. The dissociation constant (Kd) and site density (Bmax) were found to be 2.33 +/- 0.38 nM and 560 +/- 48 fmol/10(6) cells, respectively, as determined from Scatchard analysis. In competition studies for the displacement of (3H)-LTD4 from binding sites, leukotrienes B4, C4, D4 and E4, and the peptidoleukotriene antagonist FPL-55712 revealed an order of potency of LTD4 (Ki 3.9 nM) greater than LTE4 (Ki 243.9 nM) greater than LTC4 (Ki 796.9 nM) greater than FPL-55712 (Ki 17.6 microM). Concentrations of LTB4 up to 10 microM did not displace the (3H)-LTD4 binding. Bioconversion of LTD4 by GPAMs, as determined by Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC), was less than 3% in 30 min incubation periods. It is concluded that these binding sites may be receptors for LTD4 on GPAMs. Since LTD4 is produced by GPAMs, it is postulated that endogenous LTD4 may modulate thromboxane synthesis and lung constriction.     

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.     

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)     

Biosynthesis and metabolism of leukotrienes

Leukotrienes are metabolites of arachidonic acid derived from the action of 5-LO (5-lipoxygenase). The immediate product of 5-LO is LTA4 (leukotriene A4), which is enzymatically converted into either LTB4 (leukotriene B4) by LTA4 hydrolase or LTC4 (leukotriene C4) by LTC4 synthase. The regulation of leukotriene production occurs at various levels, including expression of 5-LO, translocation of 5-LO to the perinuclear region and phosphorylation to either enhance or inhibit the activity of 5-LO. Several other proteins, including cPLA2a (cytosolic phospholipase A2a) and FLAP (5-LO-activating protein) also assemble at the perinuclear region before production of LTA4. LTC4 synthase is an integral membrane protein that is present at the nuclear envelope; however, LTA4 hydrolase remains cytosolic. Biologically active LTB4 is metabolized by w-oxidation carried out by specific cytochrome P450s (CYP4F) followed by b-oxidation from the w-carboxy position and after CoA ester formation. Other specific pathways of leukotriene metabolism include the 12-hydroxydehydrogenase/15-oxo-prostaglandin-13-reductase that forms a series of conjugated diene metabolites that have been observed to be excreted into human urine. Metabolism of LTC4 occurs by sequential peptide cleavage reactions involving a g-glutamyl transpeptidase that forms LTD4 (leukotriene D4) and a membrane-bound dipeptidase that converts LTD4 into LTE4 (leukotriene E4) before w-oxidation. These metabolic transformations of the primary leukotrienes are critical for termination of their biological activity, and defects in expression of participating enzymes may be involved in specific genetic disease.     

Hyperoxic lung damage in mice: appearance and bioconversion of peptide leukotrienes

High concentrations of oxygen damage the lung and increase bronchoalveolar lavage (BAL) fluid levels of leukotrienes. We sought to identify the specific leukotrienes produced and their relationship to the severity of the lung damage and the inflammatory cell populations by exposing mice to 100% oxygen for up to 4 days. Leukotrienes were not detected in BAL fluid from air-exposed mice. Leukotriene D4 (LTD4) was found after 2 days of exposure to 100% oxygen, increased with longer periods of exposure, and then decreased while LTE4 appeared when the lung damage became severe. LTB4 and LTC4 were not found at any time. Neutropenic mice had identical results, indicating that neutrophils were not the source of the leukotrienes. To determine why LTC4 was not found and why LTD4 decreased and LTE4 increased on day 4, we measured the metabolic capacity of BAL supernatant for leukotrienes. Incubation of LTD4 in BAL supernatant from air-exposed mice resulted in the conversion of LTD4 to LTE4, which was blocked by L-cysteine, a dipeptidase inhibitor. Faster conversion occurred after exposure to 100% oxygen for 3 and 4 days. The rate of bioconversion correlated with the BAL protein concentration (r = 0.756, P less than 0.001), and it was similar in neutropenic and nonneutropenic mice. Little LTC4 and no LTE4 were converted in BAL supernatant from air- or oxygen-exposed mice. The early and progressive increase in LTD4 suggests that sulfidopeptide leukotrienes may play a role in the pathogenesis of hyperoxic lung damage. The increased dipeptidase activity during hyperoxic exposure may serve a protective role by converting the more potent LTD4 to the less potent LTE4.     

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.     

Release of leukotrienes from guinea pig lung stimulated by C5ades Arg anaphylatoxin

The complement anaphylatoxins C5a and C5Ades Arg contract guinea pig peripheral airway preparations and trachea by a mechanism largely independent of histamine release. In trachea the contractions are inhibited by FPL 55712, a relatively specific inhibitor of slow-reacting substance of anaphylaxis (SRS-A). SRS-A is now known to be a mixture of leukotrienes C4, D4, and E4 (LTC4, LTD4, LTE4). These data suggest that C5-derived anaphylatoxins stimulate production and release of leukotrienes in pulmonary tissues. To define these observations more precisely, fragments of guinea pig lung were incubated with porcine C5ades Arg, and the supernatant fluids were analyzed for leukotrienes by using both pharmacologic and chemical methods. In addition to histamine, a smooth muscle contracting activity characteristic of SRS-A was released from C5a-treated lung preparations. The contractile substance was identified as a leukotriene based on: 1) the characteristic contraction of guinea pig ileum, 2) inhibition of the contractile activity by FPL 55712, 3) enhanced release of activity in the presence of indomethacin or L-cysteine, 4) chromatographic behavior of ethanol-extracted active material on Amberlite XAD-7 resin, and 5) cochromatography of the active material on reverse-phase, high performance liquid chromatography with standard LTD4. We therefore concluded the humoral factor C5ades Arg induces a leukotriene release reaction in guinea pig lung tissue. This particular response of pulmonary tissue to anaphylatoxin has not been appreciated previously as an immediate effect of complement activation on the pathophysiology of the lung.     

Influence of hypoxia on 5-lipoxygenase pathway in rat alveolar macrophages

The effect of hypoxia was studied on the ionophore A23187-induced leukotriene production by rat alveolar macrophages. The production of LTB4 and LTC4 decreased with reducing oxygenation without change of cell viability. The synthesis of 5-HETE increased during hypoxia and the total production of LTB4, LTC4 and 5-HETE, the major metabolites of the 5-lipoxygenase pathway in rat alveolar macrophages, was equal during normoxia and hypoxia. Arachidonate release and LTA4-converting into LTB4 and LTC4 was unaffected by hypoxia. LTB4- and LTC4-degradating activities were not affected by hypoxia. These results suggest that LTA4 synthase reaction of leukotrienes biosynthesis might be suppressed by hypoxia.