Arachidonate lipoxygenase inhibitors in guinea-pig isolated trachea. Effects on contractions to antigen and various agonists

We compared the effects of the leukotriene (LT) D4 receptor antagonist FPL55712 and some lipoxygenase inhibitors on contractions of isolated guinea-pig trachea induced by antigen (ovalbumin, OA) and calcium ionophore A23187 in the presence of the cyclooxygenase inhibitor indomethacin (5 microM), and by arachidonic acid (AA), melittin and LTD4. FPL55712 (0.1 and 1 microM) inhibited contractions induced by AA (100 microM) and the phospholipase A2 activator melittin (3 micrograms/ml), while the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 microM) was a more effective inhibitor of the melittin response than the AA response. FPL55712 inhibited contractions induced by OA (100 micrograms/ml) more than by A23187 (1 microgram/ml), and these inhibitory effects of FPL55712 were much less in the presence of l-serine-borate complex (45 mM), an inhibitor of LTC4 conversion to LTD4. NDGA (10 microM) had no significant effect on the OA response, whereas the lipoxygenase inhibitors 1-phenyl-3-pyrazolidone (phenidone, 10 microM) and 5,8,11,14-eicosatetraynoic acid (ETYA, 10 microM) clearly inhibited it. In contrast, NDGA and phenidone inhibited the A23187 response, but ETYA had no effect on it. FPL55712, phenidone and ETYA, but not NDGA, had a large inhibitory effect on LTD4-induced contractions, but these inhibitors had no effect on histamine-induced contractions. These results suggest that in the guinea-pig trachea inhibitors of LTD4-induced contractions decrease antigen-induced contractions, whereas lipoxygenase inhibitors reduce the contraction to A23187.     

Inhibitory effects of a lipoxygenase inhibitor nordihydroguaiaretic acid on antagonism of leukotriene C4-induced contractions of isolated guinea-pig trachea

We have studied the effects of a lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) on antagonim of leukotriene (LT) C₄-induced contractions of isolated guinea-pig trachea and the results were compared to that of a cycloocygenase inhibitor indomethacin, NDGA (30 μM) as well as idomethacin (5 μM) inhibited LTC₄-iduced contraction. But in the presence of indomethacin NDGA was ineffective to inhibit the LTC₄ response, whereas two other lipoxygenase inhibitors, phenidone (3–30 μM) and 5,8,11,14-eicostatetraynoic acid (ETYA, 10 μM), markedly inhibited it. The antagonist action of an LTD₄ receptor antagonist FPL55712 against LTC₄-induced contractions was significantly reduced by NDGA (10–30 μM), but indomethacin had no effect on it. NDGA possessed the same inhibitory effect n the LTC₄ antagonism in the presence of indomethacin, but 0.3 μM phenidone and 1 μM ETYA which did not inhibit the LTC₄ response had no effect on it. NDGA also inhibited the relaxant response of isoproterenol on the contraction elicited by 30 nM LTC₄, but did not affect those of forskolin and aminophylline. The relaxant response of isoproterenol on the LCT₄ response was not inhibited by indomethacin, 0.3 μM phenidone and 1 μM ETYA. In the presence of a γ-glutamyltranspeptidase inhibitor, L-serine borate (SB, 45 mM), NDGA had no effect on the LTC₄ antagonism and the relaxant response of isoproterenol. In contrast, NDGA significantly inhibited the relaxant response of isoproterenol on 30 μM histamine- and 30 μM acetylcholine-induced contractions, but it did not affect the histamine antagonism by a histamine H₁-blocker pyrilamine. These results suggest that some putative nonprostanoids are involved in LTC₄-induced contractions of guinea-pig trachea and which regulate the effects of LTD₄ antagonism and β-adrenoceptor activation.     

Inhibition of leukotriene-induced contraction of guinea pig trachea by 5-lipoxygenase inhibitors

The effects of the 5-lipoxygenase inhibitors nordihydroguiaretic acid (NDGA), 5, 8, 11, 14-eicosatetraynoic acid (ETYA), 1-phenyl-3-pyrazolidone (phenidone) and BW-755c, on the contractile response to LTC4 or LTD4 were examined on the isolated guinea pig trachea. Responses to either LTC4 or LTD4 were obtained on indomethacin treated tissues, in the presence of either L-serine-borate complex or L-cysteine, respectively, to inhibit metabolic conversion of the leukotrienes. NDGA (30 microM) and ETYA (100 microM) produced a selective competitive antagonism of LTD4-induced contractions, while phenidone antagonized both LTC4- and LTD4-induced responses in a non-competitive manner. In contrast, BW-755c (30 microM) did not significantly antagonize LTC4 or LTD4 concentration-response curves. The results suggest that leukotriene antagonism may be produced by large concentrations of some 5-lipoxygenase inhibitors.     

Arachidonate lipoxygenase inhibitors in guinea-pig isolated trachea. Effects on contractions to antigen and various agonists

We compared the effects of the leukotriene (LT) D₄ receptor antagonist FPL55712 and some lipoxygenase inhibitors on contractions of isolated guinea-pig trachea induced by antigen (ovalbumin, OA) and calcium ionophore A23187 in the presence of the cyclooxygenase inhibitor indomethacin (5 μM), and by arachidonic acid (AA), melittin and LTD₄. FPL55712 (0.1 and 1 μM) inhibited contractions induced by AA (100 μM) and the phospholipase A₂ activator melittin (3 μg/ml), while the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 μM) was a more effective inhibitor of the melittin response than the response. FPL55712 inhibited contractions induced by OA (100 μg/ml) more than by A23187 (1 μg/ml), and these inhibitory effects of FPL55712 were much less in the presence of l-serine-borate complex (45 mM), an inhibitor of LTC₄ conversion to LTD₄. NDGA (10 μM) had no significant effect on the OA response, whereas the lipoxygenase inhibitors 1-phenyl-3-pyrazolidone (phenidone, 10 μM) and 5,8,11,14-eicosatetraynoic acid (ETYA, 10 μM) clearly inhibited it. In contrast, NDGA and phenidone inhibited the A23187 response, but ETYA had no effect on it. FPL55712, phenidone and ETYA, but not NDGA, had a large inhibitory effect on LTD₄-induced contractions, but these inhibitors had no effect on histamine-induced contractions. These results suggest that in the guinea-pig trachea inhibitors of LTD₄-induced contractions decrease antigen-induced contractions, whereas lipoxygenase inhibitors reduce the contraction to A23187.     

Antagonism by ETYA of the effects of leukotrienes on ileum and lung parenchymal strips independent of effects on arachidonic acid metabolism

5,8,11,14-Eicosatetraynoic acid (ETYA), a compound which inhibits both the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism, antagonized the contraction of segments of guinea-pig ileal longitudinal muscle produced by SRS-A (IC₅₀ = 2.73 μM). This activity was unaffected by pretreatment of the tissues with 10 μM indomethacin. Phenidone, another mixed cyclooxgenese-lipoxygenese inhibitor, was inactive. FPL-55712, an SRS-A antagonist, was a very potent inhibitor (IC₅₀ = 0.011 μM).BW755C and NDGA nonselectively inhibited the contractions of the guinea-pig ileal longitudinal muscle induced by SRS-A or histamine.ETYA antagonized the contraction of the guinea-pig ileal strip produced by 6 nM synthetic LTC₄ (IC₅₀ = 9.3 μM). FPL-55712 demonstrated an IC₅₀ of 0.3 μM in a similar series of experiments. ETYA, 1, 3 or 10 μM did not inhibit the contractions elicited by 0.5 μM of histamine.This was not a tissue-selective effect since 100 μM ETYA antagonized the LTC₄-induced contraction of the guinea-pig lung parenchymal strip preparation.These data demonstrate that ETYA antagonized the contractile effect of the leukotrienes on tissues from the gastrointestinal tract and lung. Furthermore, the inability of indomethacin or phenidone to inhibit the contractile response suggests that antagonism by ETYA may occur by a mechanism independent of cyclooxygenase and lipoxygenase enzymes.     

Prostaglandin F2 stimulates the generation of lipoxygenase products in guinea pig isolated trachea

The generation of lipoxygenase products on the contraction elicited by prostaglandin (PG) F2 alpha was investigated in the guinea-pig isolated trachea. Indomethacin (5 x 10(-6) M) inhibited the response at low concentrations of PGF2 alpha while enhanced the response at higher concentrations of PGF2 alpha. Phenidone (10(-4) M) and nordihydroguaiaretic acid (NDGA, 3 x 10(-5) M) appeared to inhibit the PGF2 alpha response. The PGF2 alpha response augmented by indomethacin was dose-dependently inhibited by NDGA and a leukotriene (LT) antagonist, FPL55712. NDGA had no effect on the contraction elicited by histamine but markedly inhibited the contraction elicited by LTD4. The inhibition by NDGA of the LTD4-induced contraction was abolished in the presence of indomethacin (5 x 10(-6) M). FPL55712 inhibited the LTD4-induced contraction but the extent of the antagonism was not changed by indomethacin. In the presence of indomethacin PGF2 alpha (10(-8) M) did not affect the LTD4 (3 x 10(-9) M) response but significantly enhanced the arachidonic acid (AA, 6.6 x 10(-5) M)-induced contraction. FPL55712 (3 x 10(-6) M) completely inhibited the AA response augmented by PGF2 alpha. These results suggest that lipoxygenase-mediated LT-like substances are released in the response at higher concentrations of PGF2 alpha on the guinea-pig isolated trachea, and the mode of action of PGF2 alpha is different from those of histamine and LTD4.     

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.     

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)     

Effects of immunological sensitization on the responses and sensitivity of guinea pig airways to bronchoconstrictors. Modulation by selective inhibition of arachidonic acid metabolism

The force generated by tracheal spirals and lung parenchymal strips from normal and ovalbumin-sensitized guinea pigs was measured in vitro, after challenge with histamine, carbachol, leukotriene (LT) C4, LTD4, or a prostaglandin endoperoxide analog (U-44069). The responses and sensitivity of airway tissues to the above agonists were identical in normal and sensitized animals. Treatment of tracheal spirals with indomethacin (8.5 microM), phenidone (185 microM), and nordihydroguaiaretic acid (NDGA: 30 microM) reduced resting tension (tone) equally in both normal and sensitized trachea, but did not affect lung parenchymal strips from either group. The responses of tracheal spirals from normal and sensitized animals to low concentrations of histamine, carbachol, LTC4, and LTD4 were reduced or abolished by treatment with the above inhibitors. Responses to higher concentrations of the same agonists were significantly enhanced. In contrast, treatment of normal and sensitized trachea with indomethacin (2.8 and 8.5 microM) did not abolish or reduce the effects of low concentrations of U-44069. However, an enhancement of the effect of high concentrations occurred only on normal tracheal spirals, even though the control tissues from each group responded identically with U-44069 in the absence of any inhibitor. Parenchymal strips increased in sensitivity to histamine, but not carbachol, as a result of time, vehicle, or prior exposure to the drug. Inhibitor treatment did not affect sensitivity or responsiveness of parenchyma to histamine, carbachol, and U-44069, but the contractile activity of LTD4 on both normal and sensitized lung parenchymal strips was reduced by indomethacin, NDGA, and phenidone. We conclude that ovalbumin sensitization does not induce hyperreactivity of guinea pig airways.     

Inhibition of leukotriene-induced contraction of guinea pig trachea by 5-lipoxygenase inhibitors

The effects of the 5-lipoxygenase inhibitors nordihydroguiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), 1-phenyl-3-pyrazolidone (phenidone) and BW-755c, on the contractile response to LTC₄ or LTD₄ were examined on the isolated guinea pig trachea. Responses to either LTC₄ or LTD₄ or LTD₄ were obtained on indomethacin treated tissues, in the presence of either L-serine-borate complex or L-cysteine, respectively, to exhibit metabolic conversion of the leukotrienes. NDGA (30 μM) and ETYA (100 μM) produced a selective competitive antagonism of LTD₄ - induced contractions, while phenidone antagonized both LTC₄- and LTD₄ - induced responses in a non-competitive manner. In contrast, BW-755c (30 μM) did not significantly antagonize LTC₄ or LTD₄ concentration-response curves. The results suggest that leukotriene antagonism may be produced by large concentrations of some 5-lipoxygenase inhibitors.     

Effect of indomethacin on airway contraction and the release of LTC4-like material

Ovalbumin (OA) and arachidonic acid (AA) were used to induce contractions of sensitized guinea-pig tracheal and lung preparations in the presence and absence of indomethacin. Leukotriene (LT)C4-like material released from these tissues was extracted from the bathing fluid and measured by radioimmunoassay. Challenge with either OA or AA induced release of LTC4-like material from both parenchyma and trachea, AA inducing a greater release than OA although OA induced greater contractions. This suggested that OA-induced the synthesis of other bronchoconstrictor compounds than LTC4. Although indomethacin enhanced OA- and AA-induced contractions of trachea, there was no enhancement of the release of LTC4-like material, suggesting enhancement by indomethacin was a result of the inhibition of the synthesis of prostaglandin E2 and not diversion of AA into the lipoxygenase pathway. Indomethacin had no effect on OA-induced contractions of parenchyma, but attenuated those induced by AA. Indomethacin had no modulatory effect on the release of LTC4-like material in the parenchyma. The results demonstrate that indomethacin does not result in increased synthesis of LTs in the airways.     

Arachidonic acid metabolism and casein secretion in lactating rabbit mammary epithelial cells: effects of inhibitors of prostaglandins and leukotrienes synthesis

The metabolism of arachidonic acid (AA) in fragments of lactating rabbit mammary glands in vitro was studied by considering the distribution of 13-[14C]AA in the cells, and the effects of inhibitors of cyclooxygenase and lipoxygenase pathway on the basal and prolactin (PRL)-stimulated casein secretion. 13-[14C]AA was incorporated in all classes of lipids and PRL increased transiently the percentage of free fatty acid after 1 and 5 min. Ten microM ETYA (5,8,11,14-Eicosatetraynoic acid), a tetrayne analogue of AA inhibited prostaglandins F2 alpha (PGF2 alpha) production but not leukotrienes B4 and C4 (LTB4 and LTC4) production and increased basal casein secretion. 10(-4) M DCHA (Docosahexaenoic acid) a competitive inhibitor of prostaglandin-synthetase inhibited PGF2 alpha production but did not affect basal nor PRL-stimulated casein secretion. Fourteen microM indomethacin inhibited PGF2 alpha and LTC4 production and PRL-stimulated casein secretion. Ten microM NdgA (nordihydroguaiaretic acid) an inhibitor of lipoxygenase pathway, inhibited LTB4 and LTC4 production, increased basal level of casein secretion and inhibited PRL-stimulated casein secretion. Hundred microM caffeic acid, an inhibitor of glutathione-S-transferase (GST), a class of enzymes implied in the transformation of LTA4 into LTC4, had the same effect that NDGA on basal and PRL-stimulated casein secretion. These findings show that inhibitors of AA metabolites alter casein secretion.     

Specific enhancement of LTD4-induced contractions of isolated guinea pig trachea by 5-hydroxyeicosatetraenoic acid (5-HETE)

In view of the likely production of monohydroxyeicosatetraenoic acids (HETEs) in bronchial asthma, the role of these lipoxygenase products in the development of a classical clinical element of airway disease, namely airway hyperreactivity, has been investigated. Tracheas removed from guinea-pigs actively sensitized to ovalbumin produced, upon antigenic challenge (0.01 microgram/ml), a 17-fold increase (0.97 +/- 0.34 ng/ml to 16.73 +/- 1.58 ng/ml) in the amount of 5-hydroxyeicosatetraenoic acid (5-HETE) as measured by radioimmunoassay of the tissue-bath fluid, indicating that this tissue is capable of producing 5-HETE. While 5-HETE alone, at concentrations equal to or greater than those found during the above antigenic response (0.001 to 1.0 microM), failed to produce intrinsic contractions of normal, nonsensitized guinea-pig trachea, a 30 min pretreatment with 5-HETE (1.0 microM) enhanced subsequent LTD4-induced contractions. Pretreatment with either 12- or 15-HETE, at similar concentrations and conditions, failed to potentiate LTD4 concentration-response curves. The effect of 5-HETE was time-dependent, since pretreatment for either 15 or 60 min had little or no effect on subsequent LTD4 responses. Also, the 5-HETE-induced enhancement seemed specific for LTD4, since contractions to LTC4 (in the presence of I-serine borate), acetylcholine, histamine, PGD2 or U-46619 were unaffected by 5-HETE. Therefore, 5-HETE may have a role in the development of airway hyperreactivity by interacting with released LTD4 to exacerbate airway smooth muscle contraction in asthma.     

Rat hypothalamic corticotropin-releasing hormone secretion in vitro is stimulated by interleukin-1 in an eicosanoid-dependent manner

We studied the effect of interleukin-1 alpha (IL-1) on corticotropin-releasing hormone (CRH) secretion by explanted rat hypothalami in vitro. We also assessed possible mediation of arachidonic acid metabolites on IL-1-stimulated CRH secretion, by preincubating hypothalami with the cyclooxygenase inhibitor indomethacin (INDO, 1 microM), the lipoxygenase and cyclooxygenase inhibitor eicosatetraynoic acid (ETYA, 10 microM), or the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, up to 30 microM). In additional experiments, prostaglandins (PG) E2 and F2 alpha were added to the cultures treated with INDO or ETYA. Finally, we investigated the effect of dexamethasone (DEX) on IL-1-stimulated CRH secretion. IL-1 stimulated immunoreactive CRH (iCRH) secretion by explanted hypothalami in a concentration-dependent fashion. Both INDO and ETYA inhibited IL-1-(10nM)-stimulated iCRH secretion, whereas NDGA did not have any effect. The addition of PGF2 alpha (10 nM) restored the secretion of iCRH inhibited by INDO. DEX treatment significantly inhibited IL-1-stimulated iCRH release. Our results suggest that the stimulatory effect of IL-1 on the hypothalamic CRH neuron is mediated by the cyclooxygenase metabolites of arachidonic acid, and, among others, by PGF2 alpha.     

Glucose-stimulated protein phosphorylation in the pancreatic islet

The effect of inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism on steroidogenesis in rat testis Leydig cells and rat tumour Leydig cells has been investigated. In the presence of nordihydroguaiaretic acid [NDGA; 4,4'-(2,3- dimethylbutan -1,4- diyl )bis[1,2- benzendiol ]], 5,8,11,14-eicosatetraynoic acid (ETYA), BW 755C [3-amino-1-[3-(trifluoromethyl)phenyl]-2-pyrazoline hydrochloride] and benoxaprofen [ Opren ; 2-(2-p-chlorophenyl- benzoxazol -5-yl)propionic acid)] (which inhibit lipoxygenase activity), but not indomethacin and aspirin (which inhibit cyclo-oxygenase activity), a dose-related inhibition of lutropin (LH)-stimulated testosterone and pregnenolone production was obtained (ID50 values of 2.5, 30, 25 and 30 microM for NDGA, ETYA, BW 755C and benoxaprofen were obtained, respectively). BW 755C and benoxaprofen had no significant effect on LH-stimulated cyclic AMP production except at the highest concentrations examined (330 and 380 microM, respectively), whereas NDGA and ETYA inhibited LH-stimulated cyclic AMP production in a dose-dependent manner (ID50 7.0 and 22 microM respectively). However, NDGA and ETYA also caused a dose-dependent inhibition of dibutyryl cyclic AMP-stimulated testosterone and pregnenolone production. The metabolism of exogenous ( 22R )-hydroxycholesterol or pregnenolone to testosterone by Leydig cells was not inhibited by either NDGA, ETYA or indomethacin. At low concentrations of NDGA and ETYA a significant increase in the conversion of both pregnenolone and ( 22R )-hydroxycholesterol to testosterone was obtained. Studies in which the metabolism of [14C]arachidonic acid by purified rat tumour Leydig cells was investigated indicate that products are formed by tumour Leydig cells that have similar mobilities in a thin layer chromatography system to 5-L-hydroxy-6,8,11,14-eicosatetraenoic acid, 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid and leukotriene B4. The formation of these products was inhibited to varying degrees by NDGA, BW 755C and benoxaprofen but not by aspirin and indomethacin. These studies demonstrate for the first time that inhibition of lipoxygenase activity but not cyclo-oxygenase activity causes an inhibition of LH- and dibutyryl cyclic AMP-stimulated steroid production and suggest a stimulatory role for products of the lipoxygenase pathway of arachidonic acid metabolism in steroidogenesis. The site of this stimulation is apparently distal to the production of cyclic AMP and before the side chain cleavage of cholesterol.     

Wy-48,252 (1,1,1-trifluoro-N-[3-(2-quinolinylmethoxy)phenyl]methane sulfonamide) an orally active leukotriene D4 antagonist: pharmacological characterization in vitro and in vivo in the guinea pig

The following communicates the pharmacology of Wy-48,252 (1,1,1-trifluoro-N-[3-(2-quinolinylmethoxy)phenyl]methanesulfonamide) a chemically novel and orally potent leukotriene (LT) D4 receptor antagonist. In the isolated guinea-pig trachea pretreated with indomethacin (5 microM) and L-cysteine (10 mM), Wy-48,252 antagonized TD4-induced contraction with a pKB = 7.6. Against LTC4 on tissues pretreated with IND and glutathione (10 mM), Wy-48,252 had a pKB greater than 5. Wy-48,252 (10 microM) did not antagonize pilocarpine-, histamine- or PGF2 alpha-induced tracheal contraction. Further, in the presence of indomethacin and chlorpheniramine (1 microM), Wy-48,252 dose-dependently inhibited the antigen-induced contraction of guinea-pig trachea in a manner consistent with antagonism at the LTD4 receptor and inhibition of LT synthesis. In the Konzett-Rossler model of i.v. LTD4-induced bronchoconstriction in indomethacin treated guinea pigs, intragastric Wy-48,252 (2 hr) had an ID50 of 100 micrograms/kg and a functional half-life of 5 hr. Against i.v. antigen-induced bronchoconstriction in guinea pigs treated with indomethacin and chlorpheniramine, intragastric Wy-48,252 (2 hr) had an ID50 of 0.6 mg/kg and a 5 hr half life. Intragastric Wy-48,252 also selectively blocked the cutaneous wheal reaction to intradermal LTD4 but not histamine. We conclude that Wy-48,252 is distinguished from other selective LTD4 receptor antagonists by its oral potency and should be useful in ascertaining the role of LTD4 mediated processes in asthma, allergy and animal models.     

Inhibition of arachidonic acid-induced contraction of guinea pig lung strips

The effects of several enzyme inhibitors on arachidonic acid-induced contractions of guinea pig lung strips were studied. Varying concentrations of indomethacin, an inhibitor of cyclooxygenase, produced only a limited effect on contraction of tissue strips. By contrast, nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), and phenidone, which inhibit either lipoxygenase, or both lipoxygenase and cyclooxygenase, caused a dose-related antgonism of the arachidonic acid-induced contraction. The effects of these latter agents were similar to that of FPL 55712. Results indicate that the products of cyclooxygenase are predominantly involved in the early phase and the products of lipoxygenase are predominantly related to the late phase of arachidonic acid-induced contraction.     

Dependency of the Paf-acether induced bronchospasm on the lipoxygenase pathway in the guinea-pig

The Paf-acether (platelet-activating factor) induced bronchospasm (Paf-BCS) was studied in the anesthetized guinea-pig. The SRS antagonist, FPL-55712, as well as inhibitors of both lipoxygenase and cyclooxygenase, phenidone, nordihydroguaiaretic acid (NDGA), and benoxaprofen, caused a dose-related antagonism of Paf-BCS. By contrast, selective inhibitors of cyclooxygenase, indomethacin and aspirin, exerted moderate antagonism at intermediate doses, but had no effect at high doses. Furthermore, diethylmaleate (DEM), which impairs leukotriene synthesis by interfering with glutathione (GSH), suppressed Paf-BCS. Taken together, these results demonstrate that the lipoxygenase pathway plays a major part in the bronchospasmogenic effect of Paf-acether in the guinea-pig.     

Differentiation of the mechanisms by which leukotrienes C4 and D4 elicit contraction of the guinea pig trachea

The contractions elicited by leukotriene (LT) C4 and D4 in isolated guinea pig trachea were characterized under conditions in which LTC4 to LTD4 metabolism was blocked by the presence of 45 mM l-serine-borate complex (SB). The presence of SB caused a shift of the LTC4-concentration-response curve to the left by 7.5-fold, and blocked the bioconversion of LTC4 to LTD4 by the trachea as estimated by HPLC analysis of the LTs present in the tissue bath fluid. The potency of FPL 55712 as an antagonist of the LTC4-induced contractions in the presence of SB was 15-30-fold less than its potency as an antagonist of the LTD4-induced contractions. In contrast, another LT antagonist, SK&F 101132, equally antagonized the contractions elicited by LTC4 and LTD4 in either the presence or absence of SB. The differential antagonism of LTC4 and LTD4 implies the existence of multiple pharmacologic receptors for the LTs. The calcium channel entry blockers, nifedipine and verapamil, at concentrations as high as 10 microM, suppressed the maximal LTC4-induced contraction by no more than 20%, whereas the purported intracellular calcium antagonist, TMB-8, completely suppressed the LTC4 concentration-response curve in the presence of SB, a profile identical to that previously reported for LTD4. Thus, if multiple LT receptors exist, they appear to mobilize calcium in a qualitatively similar fashion following LT stimulation.     

Influence of platelet-activating factor on leukotriene D4-induced contractions of the guinea pig parenchymal strip

Platelet-activating factor (PAF) and sulphidopeptide leukotrienes, such as leukotriene D4 (LTD4), are potent constrictors that are probably released simultaneously in a variety of inflammatory respiratory events. The purpose of the present study was to determine whether LTD4-induced contractions of guinea pig parenchymal lung strips (GPPS) are modified in the presence of PAF. The contractile responses of isolated GPPS to cumulative doses of LTD4, acetylcholine, histamine, and potassium chloride in the presence of PAF (0.1 nM, 0.1 microM) were compared with parallel controls. There was no significant alteration of the response to acetylcholine and potassium chloride and the PAF-induced inhibition of the response to histamine, although significant, was not concentration dependent. In contrast, PAF in a concentration range from 0.1 nM to 1.0 microM caused a marked, concentration-dependent reduction of LTD4-induced contractions. Pretreatment with the PAF receptor antagonist, BN52021, prevented the attenuation of LTD4-induced contraction by PAF. The attenuation of LTD4-induced contraction by PAF was also prevented by pretreatment with indomethacin or with the thromboxane synthase inhibitor U63,557A, but not by pretreatment with the lipoxygenase inhibitors BW755c or nordihydroguaiaretic acid. Thus inhibition of LTD4-induced GPPS contraction by PAF is receptor dependent and probably secondary to thromboxane generation. The respiratory smooth muscle response to leukotrienes may be modified significantly by concomitant PAF release.