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.     

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.     

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 LTC₄, LTD₄, LTE₄, 5-hydroxyeicosatetraenoic acid (5-HETE), PGD₂, PGE₁, PGF_2α, PGI₂, 6-oxo-PGF_1α, 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 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 (1.9μM) and, when given repeatedly, caused tachyphylaxis in GPP,LTB₄ was considerable more active on GPP than the other substances investigated. Further, PGD₂, PGF_2α and PGI₂ contracted GPP, the order of potency being PGD₂ > PGF_2α ? 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-PGF_1α were inactive on both GPP and GPISM.On the basis of differential effects of LTB₄ on GPP and GPISM, this assay repressents a simple and selective means to distinguish LTB₄-like materials from other naturally-occuring substances likely to be generated in inflammatory fluids.     

Pharmacological study of the effects of leukotrienes C4, D4, E4 & F4 on guinea pig trachelis: Interaction with FPL-55712

Leukotrienes D₄ ? C₄ > E₄ ? F₄ produced qualitatively similar contractions of guinea-pig trachealis, which were antagonized by the SRS-antagonist FPL-55712. Schild analyses indicated that FPL-55712 when tested in a low concentration range (0.57–5.7 × 10^?6M) competitive antagonist of LTC₄, LTE₄ and LTF₄ (slope not significantly different from one). The interaction of FPL-55712 with LTD₄ may be noncompetitive (slope < 1). Comparison of the calculated dissociation constants (?log K_B) indicated that FPL-55712 was more effective at blocking LTE₄ and LTF₄ compared to LTC₄ and LTD₄. In the presence of higher concentrations of FPL-55712 (1.9 × 10^?5M) the antagonism of LTC₄ became noncompetitive. These findings indicate that important differences exist in the interaction of FPL-55712 with the various peptido leukotrienes in guinea pig trachealis. Discovery of more selective antagonists will be needed to determine if multiple receptor subtypes are present in this tissue.     

Leukotriene B4 and its action with a free-radical-generating system

The concentration of Leukotriene B₄ (LTB₄) demonstrated in early inflammation has been shown to induce leukocyte aggregation, chemotaxis and degranulation of polymorphonuclear leukocytes (PMN) $\text{in vitro}$. N-f-Met. Leu-Phe, a potent chemotactic factor, has been shown to activate neutrophils to produce chemiluminescence and produce superoxide radicals. The characteristics of the LTB₄-induced degranulation of rabbit neutrophils are strikingly similar to those of the chemotactic factors. Thiols, and in partiicular glutathione, have been shown to have a marked inhibitory effect in clinical assays of superoxide dismutase (SOD) activity, using reactions which are supposedly specific for the superoxide ion. SOD is most frequently assessed by coupling a generator of O₂^? with an indicating scavenger for the radical. The enzyme then competes with the scavenger for the available O₂^? and inhibits the processes being observed, thus, the inhibition serves as a basis for estimation of SOD activity. A method proposed by Misra and Fridovich for the estimation of SOD activity is based on the photo-oxidation of dianisidine sensitised by riboflavin.This assay can be used to classify compounds as either SOD-like or glutathione-like. With a small quantity if LTB₄ and LTD₄, we obtained preliminary results for their effect on the assay (Table 1). They appear to be glutathione-like, i.e., reactive with the free-radical-generating system in preference to a specific reaction with O₂^? and are only slightly less effective than glutathione.Although our results are preliminary it is clear that the leukotrienes are effective as radical scavengers in this reaction. Further studies with two prostaglandis (products of the cycloxygenase pathway) will also be presented.     

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.     

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 substances (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. Indo-methacin (1 μg/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 on the GPP was abolished by FPL 55712 (0.5 – 1.0 μg/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 μg/ml failed to antagonise leukotriene-induced contractions.     

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.     

Thromboxane A2 antagonist inhibits leukotriene D4-induced smooth muscle contraction in guinea-pig lung parenchyma, but not in trachea

Although the bronchoconstriction induced by leukotriene D₄ (LTD₄) has been reported to be partly mediated by thromboxane A₂ (TXA₂) in the guinea-pig airway, it is not known which part of the airway is susceptible to TXA₂. In order to determine the role of TXA₂ in the central and peripheral airways, we compared the effect of a TXA₂ antagonist on tracheal strips to its effect on parenchymal strips of guinea-pigs. Tracheal and parenchymal strips were mounted in a 3.5 ml organ bath filled with Krebs-Henseleit solution aerated with 95% O₂, 5% CO₂ and kept at 37°C. After equilibration for 60 min in Krebs solution, the strip was contracted by exposure to 10⁻⁵ M of acetylcholine (ACh). Sixty minutes after ACh was eliminated, the concentration-response curve to LTD₄ (10⁻⁹ M–10⁻⁷ M) was obtained, and the LTD₄-induced contractions were expressed as the percent of the contraction evoked by 10⁻⁵ M of ACh. We measured the contractile response to LTD₄ in the presence or absence of the TXA₂ antagonist, BAY u3405 (10⁻⁸ M–10⁻⁶ M). In the tracheal strips, BAY u3405 had no effect on the LTD₄-induced contraction. However, in parenchymal strips, BAY u3405 significantly suppressed the contractile response to LTD₄. These results suggest that in the central airway LTD₄ contracts smooth muscle directly, but that in the peripheral airway LTD₄ induces smooth muscle contraction both directly and indirectly, via TXA₂.     

Pulmonary microcirculatory responses to leukotrienes B4, C4 and D4 in sheep

The pulmonary microvascular responses to leukotrienes B₄, C₄ and D₄ (total dosage of 4 μg/kg i.v.) were examined in acutely-prepared halothane anesthetized and awake sheep prepared with lung lymp fistulas. In anesthetized as well as unanesthetized sheep, LTB₄ caused a marked and transient decrease in the circulating leukocyte count. Pulmonary transvascular protein clearance (pulmonary lymph flow x lymph-to-plasma protein concentration ratio) increased transiently in awake sheep, suggesting a small increase in pulmonary vascular permeability. The mean pulmonary artery pressure (P ) also increased. In the acutely-prepared sheep, the LTB₄-induced pulmonary hemodynamic and lymph flow responses were damped. Leukotriene C₄ increased P to a greater extent in awake sheep than in anesthetized sheep, but did not significantly affect the pulmonary lymph flow rate (Q̇_lym) and lmph-to-plasma protein concentration (L/P) ration in either group. LTD₄ increased P and Q̇_lymp in both acute and awake sheep; Q̇_lym increased without a significant change in the L/P ratio. The LTD₄-induced rise in P occurred in association with an increase in plasma thromboxane B₂ (Txb₂) cocentration. The relativity small increase in Q̇_lym with LTD₄ suggests that the increase in the transvascular fluid filtration rate is the result of a rise in the pulmonary capillary hydrostatic pressure. In conclusion, LTB₄ induces a marked neutropenia, pulmonary hypertension, and may transiently increase lung vascular permeability. Both LTC₄ and LTD₄ cause a similar degree of pulmonary hypertension in awake sheep, but had different lymph flow responses which may be due to pulmonary vasoconstriction at different sites, i.e. greather pre-capillary constriction with LTC₄ because Q̇_lym did not change and greater post-capillary constriction with LTD₄ because Q̇ increased with the same rise in P .     

The pharmacological characterization of 5-HT3 receptor binding sites in rabbit ileum: Comparison with those in rat ileum and rat brain

We have further characterized the 5-HT₃ receptors in rat and rabbit tissues by evaluating the binding of the 5-HT₃ receptor ligand, [³H]GR67330 to homogenates of rabbit ileum, rat ileum and rat brain (entorhinal cortex). In each tissue specific [³H]GR67330 binding represented a single saturable, high affinity site (K_d = 0.14, 0.18, 0.076 nM in rabbit ileum, rat ileum and rat brain respectively). The densities of sites present in rabbit and rat ileum were similar to that present in rat brain (B_max = 63, 47, 72 fmol/mg protein in rabbit ileum, rat ileum and rat brain respectively).

In each tissue, 5-HT₃ receptor agonists and antagonists potently competed for [³H]GR67330 binding. Derived inhibition constants were similar in rat ileum and brain. However marked differences in IC₅₀s were apparent for rabbit ileum compared with rat brain or ileum. These were most apparent with agonists. Thus, mCPBG [1-(meta-chlorophenylbiguanide)], phenylbiguanide, 5-HT and 2-methyl 5-HT were at least 5 times less potent to inhibit [³H]GR67330 binding in rabbit ileum than rat brain. The most pronounced differences were evident with phenylbiguanide and mCPBG which were 70 and 300 times less potent in the rabbit ileum respectively compared with the rat tissues. These differences were unlikely to be due to depletion effects because tissue combination experiments (rabbit ileum and rat brain) yielded biphasic inhibition curves for phenylbiguanide with affinities for each component similar to those in the individual tissues. Antagonist affinities also varied between the rabbit and rat tissues, although less markedly. Amongst the antagonists, the most marked differences were apparent with SDZ 206–830 and quipazine each being 10 times less potent to inhibit binding to rabbit than rat tissue.

Hill coefficients for inhibition of binding varied with tissue. In rat brain, as previously described for [³H]GR67330, Hill coefficients for agonist (and quipazine) inhibition of binding were greater than unity. This was less marked in rat and rabbit ileum tissues.

The present studies provide further evidence for species variation in 5-HT₃ receptors.     

Binding of leukotrienes C4 and D4 to membranes from guinea pig lung: Regulation by ions and gaunine nucleotides

Tritium-labeled leukotrienes C₄ and D₄ (LTC₄ and LTD₄) bind to membranes from guinea pig lung. Binding properties of the two ligands are almost identical. More than 80% of ³H-LTC₄ and ³H- LTD₄ binding can be blocked by unlabeled LTC₄ (IC₅₀ 8 nM versus ³H-LTC₄ and 8 nM versus ³H-LTD₄), LTD₄ (12 nM, 16 nM), LTE₄ (40 nM, 98 nM), and the leukotriene antagonist FPL 55712 (14 μM, 11 μM). Binding is reversible (50% dissociation at 65 min for both ligands at 25°). Binding of ³H-LTC₄ and ³H-LTD₄ is enhanced by divalent cations and inhibited by sodium ions, guanine nucleotides, and EDTA. ³H-LTD₄ binds in unaltered form, but ³H-LTC₄ appears to bind mostly after conversion to ³H-LTD₄. The high affinity, reversibility, and regulation by ions and guanine nucleotides of ³H-LTC₄ and ³H-LTD₄ binding strongly imply that these binding sites are physiological LTD₄ receptors.     

The actions of leukotrienes C4 and D4 in the porcine renal vascular bed

The kidney of anaesthetised pigs was perfused $\text{in situ}$ with carotid arterial blood. Renal blood flow and perfusion pressure were recorded. Close intra-arterial injection of leukotriene (LT) C₄, D₄ or noradrenaline (NA) caused a dose-related increase in vascular resistance. Both LTs were more active than NA by one to two orders of magnitude. Systemically-administered indomethacin potentiated the effect of all three agonists. Incubation of renal artery tissue with calcium ionophore A23187 in the presence of indomethacin resulted in the generation of LT-like material which, when assayed on guinea-pig ileum, was indistinguishable from LTD₄. The results show that pig renal vessels produce LT-like material and suggest that the potent vasoconstriction induced by exogenous NA and LTs is modulated $\text{in vivo}$ by a vasodilator cyclo-oxygenase product.     

Vasoconstrictor effects of leukotrienes C4 and D4 in the feline mesenteric vascular bed

The effects of leukotriene C₄ (LTC₄) and leukotriene D₄ (LTD₄) in the feline mesenteric vascular bed were investigated under conditions of controlled blood flow so that changes in perfusion pressure directly reflect changes in vascular resistance. Intra-arterial injections of LTC₄ and LTD₄ (0.3–3.0 μg) increased perfusion pressure in a dose-related fashion. Vasoconstrictor responses to LTC₄ and LTD₄ were similar to norepinephrine (NE) whereas mesenteric vasoconstrictor response to the thromboxane analog, U46619, was markedly greater than were responses to LTC₄ and LTD₄. Meclofenamate in a dose that greatly attenuated the systemic depressor response to arachidonic acid was without effect on vasoconstrictor responses to LTC₄ and LTD₄, NE and U46619 in the mesenteric vascular bed. The present data show that LTC₄ and LTD₄ possess significant vasoconstrictor activity in the feline mesenteric vascular bed. In addition, the present data suggest that products of the cyclooxygenase pathway do not mediate vasoconstrictor responses to LTC₄ and LTD₄ in the intestinal circulation of the cat.     

Inflammation and pain sensitivity: Effects of leukotrienes D4, B4 and prostaglandin E1 in the rat paw

Leukotrienes (LT's) and prostaglandins (PG's) have been proposed as mediators of vascular permeability change in inflammatory reactions. Also, prostaglandins, especially of the E-type, have been shown to enhance pain responses. In the present studies in rat, the effects of LTB₄ and LTD₄ on edema and pain thresholds were examined in combination with PGE₁ and/or brewer's yeast. Subplantar injections of LTD₄ or LTB₄ induced small increases in paw thickness which were potentiated by the co-administration of PGE₁. LTD₄ alone had no significant effect on the development of the yeast paw edema. LTB₄ was found to reduce significantly the yeast edema and this reduction could be reserved by administration PGE₁. A small but significant decrease in pain threshold was caused by PGE₁ and this was significant enhanced in the presence of LTD₄. LTB₄, like PGE₁, was found to cause slight hyperalgesia but no synergy between the two agents was observed. LTD₄ was found to have no effect on the initial hypoalgesia or subsequent development of hyperalgesia caused by brewer's yeast. Both LTB₄ and PGE₁, however, prevented the initial hypoalgesia and significantly reduced tha latency for development of yeast induced hyperalgesia. These effects of LTB₄ are discussed in terms of possible release of cyclooxygenase products.     

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.     

Leukotriene C4 Transport and Metabolism in the Central Nervous System

The transport and metabolism of radiolabeled leukotriene (LT) C4 in the CNS were investigated after intraventricular injection. Under thiopental (Pentothal) anesthesia, New Zealand white rabbits were injected intracerebroventricularly with 0.2 ml of artificial CSF containing 2.5 microCi of [3H]LTC4 (36 Ci/mmol), 0.3 microCi of [14C]mannitol, and, in some cases, 0.9 mg of probenecid, 1.8 mg of cysteine, 1.4 micrograms of unlabeled LTC4, or 2 mg of tolazoline HCl. After 2 h, the conscious rabbits were killed, and the quantity and nature of the 3H and 14C were determined in CSF, choroid plexus, and brain. The [3H]LTC4 recovered in CSF and brain was not extensively metabolized, as greater than 70% of the 3H remained [3H]LTC4, although some spontaneous conversion to 11-trans-[3H]LTC4 occurred. Oxidized forms of [3H]LTC4, [3H]LTD4, and [3H]LTE4 did not exceed 18% in CSF and brain. After intraventricular injection of [3H]LTC4, 3H was transferred from the CSF to blood by a probenecid-sensitive, but tolazoline-insensitive, transport system in the CNS much more rapidly than mannitol. Cysteine decreased the retention of [3H]LTC4 in brain. These results are consistent with previous in vitro observations that [3H]LTC4 is transferred from CSF into blood by an efficient transport system for LTC4 in choroid plexus.     

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.