Guinea Pig Lung Parenchymal Strips Research Articles

Substituted arylmethyl phenyl ethers. 1. A novel series of 5-lipoxygenase inhibitors and leukotriene antagonists

A series of new substituted arylmethyl phenyl ethers has been prepared. These compounds were tested as inhibitors of 5-lipoxygenase (5-LO) in rat neutrophils, in vitro antagonists of leukotriene-induced contraction of guinea pig (GP) lung parenchymal strips, and inhibitors of slow reacting substance of anaphylaxis (SRS-A) mediated bronchospasm in the GP in vivo. Most representatives of this new class of potential antiallergic/antiinflammatory agents showed potent inhibition of 5-LO activity in rat PMNs. The most potent compound, 2-[[3-(1-hydroxyhexyl)phenoxy]-methyl]quinoline (33), had an I50 of 0.12 microM in the rat PMN 5-LO assay and an I50 of 3.6 microM in the leukotriene-induced contraction of GP lung parenchymal strips, and it also showed 91% inhibition of SRS-A-mediated bronchospasm in the GP in vivo at 10 mg/kg, administered intraduodenally. Some of the compounds in this series were also leukotriene antagonists in vitro, and several of them showed in vivo activity against SRS-A-mediated bronchospasm in the GP.

Endothelial cell leukotriene C4 synthesis results from intercellular transfer of leukotriene A4 synthesized by polymorphonuclear leukocytes.

Leukotriene (LT) synthesis and metabolism were studied in porcine aortic endothelial cells. Leukotrienes were identified by combinations of guinea pig lung parenchymal strip bioassay, radioimmunoassay, and UV spectrophotometry with high performance liquid chromatography. Endothelial cells stimulated with the calcium ionophore, A23187, were unable to convert arachidonic acid to detectable levels of LTA4-derived products including the biologically active metabolites, LTB4 or LTC4. However, these cells readily converted exogenous LTA4 to the potent slow-reacting substance, LTC4. Smaller quantities of 11-trans-LTC4 and LTD4 were also observed. LTB4 was not detectable in these incubations nor was LTB4 metabolism observed. The possible intercellular transfer of LTA4 between polymorphonuclear leukocytes (PMNL) and endothelial cells was tested since PMNL release LTA4 when stimulated and have significant contact with endothelium. When A23187-stimulated neutrophils were coincubated with endothelial cells, a significant increase in LTC4 levels was detected over PMNL alone. LTC4 is formed by the enzymatic conjugation of glutathione (GSH) with LTA4. Therefore in some experiments, endothelial cells were prelabeled with [35S]cysteine to allow intracellular synthesis of [35S]GSH. When unlabeled PMNL were added, as a source of LTA4 to the prelabeled endothelial cells, substantial levels of [35S] LTC4 were recovered. The data indicate that endothelial cells synthesize LTC4 from LTA4. They also demonstrate a specific PMNL-endothelial cell interaction in which endothelial cell LTC4 synthesis results from the intercellular transfer of LTA4 produced by PMNL.

Characterization of platelet activating factor (PAF)-acether-induced contractions of guinea-pig lung strips by selected inhibitors of arachidonic acid metabolism and by PAF-acether antagonists

The myotropic activities of PAF-acether, leukotriene B4, leukotriene D4 and histamine were compared on superfused guinea-pig lung parenchymal strip and were shown to have the following order of potenc3: PAF-acether > LTD4 > LTB4 > histamine. The contractile response of the lung parenchyma to PAF-acether was inhibited by aspirin, imidazole and OKY-046, which suggested that thromboxane A2 might play a mediator role in PAF-induced contractions. Neither an antagonist of leukotriene D4, FPL-55712, nor an antihistamine, mepyramine, had any effect of PAF contractions. The activity of a novel antagonist of PAF-acether, BN 52021, was also studied on superfused lung parenchyma contracted by histamine, leukotriene B4, leukotrine D4 and PAF-acether. This compound was without effect on the histamine response but it slightly reduced the contractions elicited by leukotriene D4 and potentiated those by leukotriene B4. BN 52021 (7.1 × 10−6 M) inhibited by 63% the contraction induced by 5.7 × 10−13 M PAF-acether and by 52% that induced by 5.7 × 10−10 M PAF-acether and kadsurenone (8.4 × 10−6 M), another PAF-acether antagonist, inhibited the same PAF-induced contractions by 75% and 20% respectively.

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 (IC 50 = 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 (IC 50 = 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 LTC 4 (IC 50 = 9.3 μM). FPL-55712 demonstrated an IC 50 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 LTC 4-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.

Comparative effects of drugs on leukotrienes-, PAF-acether- and histamine- induced contractions of Guinea-pig lung parenchymal strips

Dose-response curves to leukotrienes B 4 (LTB 4), D 4 (LTD 4), PAF-acether and histamine applied as single or cumulative doses are studied on guinea-pig lung parenchymal strips (GPLP). A mole to mole comparison shows that PAF-acether is the most potent agonist, when GPLP contracts maximally to histamine. GPLP possesses specific receptors to histamine, LTD 4 and PAF-acether which can be blocked by selective receptors antagonists: mepyramine as antihistaminic, FPL 55712 as anti-SRS-A substance, BN 52021 and kadsurenone as anti-PAF-receptor antagonists; meanwhile, a part of their action is mediated through the release of arachidonate metabolites. Histamine, LTB 4, LTD 4 and PAF-acether-induced contractions are all dependent on cyclooxygenase. Thromboxane A 2 (TXA 2) could be one of the cyclooxygenase products involved. A part of contractile effects of LTB 4 and LTD 4, but not of histamine, is linked to the generation of lipoxygenase metabolites as their contractions are partially inhibited by NDGA and ETYA. Contraction-induced by PAF-acether is reduced in presence of NDGA but not by FPL 55712, which indicates that lipoxygenase products other than peptidoleukotrienes are involved in the PAF-response.

Sotalol potentiates the leukotriene C 4-induced contractions and thromboxane A 2 release of guinea pig lung parenchymal strips

1. 1. The leukotriene C 4 (LTC 4)-induced contraction and thromboxane A 2 (TxA 2) release of the guinea pig lung parenchymal (GPLP) strip are both inhibited by the β 2-adrenergic agent salbutamol. The effect of LTC 4 is restored to nearly normal by the β-adrenergic antagonist sotalol. 2. 2. The latter substance alone also induces a contraction in the GPLP strip and potentiates the contractions and the TxA 2 release of LTC 4. During the sotalol-induced contractions, no TxA 2 release occurs. An antihistaminic, mepyramine had no effect on the sotalol-induced contraction. 3. 3. When sotalol is added repeatedly to a GPLP strip, only the first time a contraction occurs.

Inhibition of the platelet activating factor mediated component of guinea pig anaphylaxis by receptor antagonists.

The role of platelet activating factor (PAF) and its inhibition by specific receptor antagonists was studied in hypersensitivity reactions in guinea pig lung parenchymal strips and in guinea pigs in vivo. Immunological challenge of isolated lung parenchymal strips with ovalbumin resulted in a contractile response of 184 +/- 16 mg (n = 38). Pretreatment of the strips with a combination of the antihistamine diphenhydramine, the dual lipoxygenase and cyclooxygenase product synthesis inhibitor BW-755C, and the PAF receptor antagonist kadsurenone totally inhibited the increase in tension. The bronchoconstrictor effects induced by immunological challenge were also totally inhibited when the leukotriene receptor antagonist FPL-55,712 was used to replace BW-755C or another PAF receptor antagonist, alprazolam was used to replace kadsurenone. Ovalbumin challenge in sensitized guinea pigs in vivo resulted in airway constriction, circulatory failure and an abrupt fall in continuously measured platelet count by 75 +/- 12%, followed by death of all animals within 5-8 min. Pretreatment with diphenhydramine, BW-755C and kadsurenone (or alprazolam) improved survival to 64% (i.e., 7 of 11 animals) compared to a survival rate of 0% (i.e., 0 of 8 vehicle treated controls). Despite the improved survival, the severe thrombocytopenia was unaltered with the combined therapy. PAF, histamine and peptide leukotrienes appear to act in concert in mediating the anaphylaxis-induced airway constriction and circulatory failure in guinea pigs, but are not apparently responsible for the accompanying thrombocytopenia.

Immunopharmacology of Anaphylatoxin-Induced Bronchoconstrictor Responses

The complement anaphylatoxin peptides, C3a and C5a, are potential mediators of immediate hypersensitivity reactions, eliciting many of the same actions on isolated tissue and cell preparations as specific antigen. Instilled intratracheally in experimental animals, the peptides induce acute bronchospasms and are sometimes lethal. In vitro, they cause dose-dependent contraction of isolated lung tissue preparations, a response which correlates well with bronchospasms observed in vivo, and our current understanding of the cellular and molecular mechanisms of this action are reviewed here. C5a and its catabolic derivative, C5ades Arg, stimulate contraction of isolated guinea pig lung parenchymal strips in part by production of leukotrienes that constitute SRS-A, and by release of histamine. Leukotrienes in turn release thromboxane from lung tissue, and evidence indicates that at least part of the spasmogenic activity of these peptidolipids is mediated by this effect. C3a is considerably less potent than C5a in contracting lung tissues and appears to act primarily by causing the release of spasmogenic cyclooxygenase metabolites. Both peptides may additionally have direct action on contractile cells within the tissue. Platelet-activating factor (PAF), an unusual phospholipid mediator released from inflammatory cells stimulated with C5a and other agents, also contracts isolated lung parenchymal tissues. PAF stimulates release of significant quantities of thromboxane from guinea pig lung; however, indomethacin does not block contractile responses of the tissue. Recent evidence indicates that PAF may act on parasympathetic neurons in lung to release endogenous acetylcholine, and this action may be a major component of tissue responses to this mediator. Thus the complement anaphylatoxins stimulate release of many of the same mediators from lung tissues as are released by antigen challenge of sensitized tissue, and may, therefore, play an important role in the pathogenesis of allergic bronchospasms.

Pharmacological characterization of the effects of verapamil and nifedipine on isolated guinea-pig lung parenchymal strip

1. 1. Verapamil and nifedipine elicited dose-related relaxations and produced a right downward shift of the concentration-response curves to CaCl 2, KCl, acetylcholine and histamine in the isolated guinea-pig lung parenchymal strip. 2. 2. These Ca-entry blockers had greater inhibitory effects on contractions evoked by CaCl 2 and KCl than against those elicited by acetylcholine and histamine.

IgG vs IgE: Mediators of antigen-induced guinea pig lung parenchymal contraction

The present studies were conducted to determine whether different mediators are responsible for antigen-induced contraction of guinea pig lung parenchymal strips sensitized with either IgG or IgE. Passive sensitization was employed using IgG- or IgE-type antibody to ovalbumin which had been separated using Protein A-Sepharose as previously described (Regal, 1984a). Such a system ensures the presence of only one type of cytophilic antibody to mediate antigen-induced contraction of the isolated lung parenchymal strip. Pyrilamine, an H1 antagonist, caused a very slight delay in the onset of the antigen-induced contractions in both IgG- and IgE-sensitized strips, suggesting a very minor role for endogenous histamine in both systems. A combination of the leukotriene antagonists, FPL 55712, and pyrilamine caused a significant reduction in the antigen-induced contraction in IgE- but not IgE-sensitized lung parenchymal strips. Histamine release was detected on antigen challenge of both IgG- and IgE-sensitized lung. Using bioassay techniques leukotriene release was primarily detected from IgG-sensitized lung and only minimally from IgE-sensitized lung. Thus, our studies have demonstrated a differential antagonism of IgG- and IgE-mediated airway contraction and suggest that leukotrienes are important mediators of antigen-induced lung parenchymal contraction in IgG- but not IgE-sensitized tissues.

Phorbol myristate acetate causes in guinea-pig lung parenchymal strip a maintained spasm which is relatively resistant to isoprenaline

The effect of phorbol myristate acetate (PMA) was compared with that of histamine on the guinea-pig lung parenchymal strip. PMA, 10 −5 M, caused a slowly developing sustained contraction which had approximately the same magnitude as the maximal histamine contraction. Isoprenaline, at 10 −5 M, caused 86% relaxation of the histamine contraction but only 22% relaxation of the PMA contraction. Forskolin, at 10 −5 M had a similar action to isoprenaline on the effects of both spasmogens while aminophylline, 5 × 10 −4 M, was considerably less effective. Sodium nitroprusside had little effect on the histamine contraction and actually increased the PMA spasm. It is suggested that protein kinase C may have a role in the tonic phase of the contraction of bronchiolar smooth muscle. These findings could have relevance for the delayed phase of asthma, which is known to be insensitive to β-agonists.

A study of the histamine H2-receptor mediating relaxation of the parenchymal lung strip preparation of the guinea-pig.

The relaxation produced by several H2-receptor agonists and forskolin was investigated on strips of guinea-pig lung parenchyma. Dimparit, 1 microM to 10 mM, 4-methyl histamine, 0.5 microM to 100 microM and impromidine, 10 nM to 1 microM, had no effect on the tone of the unstimulated strips of lung parenchyma but caused a dose-dependent relaxation of strips that were contracted by 2-pyridylethylamine (2-PEA), 15 microM. Forskolin, 10 nM to 4 microM, produced a dose-dependent relaxation of both the stimulated and unstimulated lung strips. The muscarinic antagonist atropine, 1 microM, and the beta 2-adrenoceptor antagonist propranolol, 10 microM, had no effect on the dose-response curve for dimaprit-induced relaxation of the lung strip. The dose-response curve for dimaprit was shifted to the right in a dose-dependent manner by increasing concentrations of a variety of H2-antagonists. Schild plots produced a straight line for all the H2-antagonists with slopes not significantly different from unity. The equilibrium dissociation constants for the H2-antagonists on the lung strip preparation were similar to those previously reported for inhibition of the chronotropic activity of histamine on guinea-pig right atria and inhibition of [3H]-tiotidine binding to homogenates of guinea-pig lung parenchyma.

Bronchorelaxing Activity of Lithium In Vitro

The potential value of lithium as a bronchodilator has been evaluated by in vitro methods using guinea pig tracheal spirals and lung parenchymal strips. The use of twin spirals and strips prepared by splitting the tissues longitudinally into two equal halves has been used to minimize animal variations. The cumulative concentration–effect relationships of isoproterenol have been determined on these twin tissues contracted submaximally with histamine. The responses of the twin tissues did not significantly differ. The cumulative concentration–effect relationships of lithium on guinea pig tracheal spirals and lung parenchymal strips have been evaluated and compared with those of isoproterenol. Lithium showed a dose-dependent relaxation on both tissues. However, the potency of lithium to produce a relaxant effect was significantly lower than isoproterenol. The relaxing effect of lithium on parenchyma was significantly higher than that on the trachea. The lithium-induced inhibitory effect on the contractile responses of tracheal spirals and lung parenchymal strips to histamine before and after incubating the tissues with lithium for 15min has been determined. Lithium significantly reduced the contractile effect of histamine on both tissues. These findings suggest a potential clinical applicability of lithium for treating airway hyperresponsiveness.

Correlation between the myotropic activity of leukotriene A 4 on guinea-pig lung, trachea and ileum and its biotransformation in situ

Leukotrienes A 4 and D 4 displayed equivalent myotropic activity on guinea pig lung parenchyma strips. However, on the trachea, the activity of LTD 4 was much higher than that of LTA 4. The potencies of these two leukotrienes were also different on strips of longitudinal muscles of the ileum where LTD 4 was very active whereas LTA 4 was inactive. Since the activities of both leukotrienes were blocked by FPL-55712, our results suggested that the transformation of LTA 4 by the smooth muscle preparations was a prerequisite to its biological activity. LTA 4 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 LTA 4 to LTB 4, LTC 4, LTD 4 and LTE 4. Incubation of LTA 4 with homogenates of trachea or of the longitudinal muscles of ileum showed the formation of LTB 4 and its isomers but no significant amount of peptido-leukotrienes were detected. These findings reveal that LTA 4 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 LTA 4 is related to the tissue levels of enzymes which catalyse its biotransformation.

Leusotriene D 4 and phospholipase A 2 release thromboxane A 2 of different origin in the guinea pig lung parenchymal strip

Leusotriene D 4 and phospholipase A 2 release thromboxane A 2 of different origin in the guinea pig lung parenchymal strip

Evidence for a role of the amino-terminal region in the biological activity of the classical anaphylatoxin, porcine C5a des-Arg-74.

The presence of methionyl residues at positions 1 and 17 in porcine classical anaphylatoxin (e.g. C5a des-Arg-74) permits chemical cleavage of the factor with cyanogen bromide to generate two defined fragments. Peptides corresponding to the amino-terminal fragment, CN-I, and the carboxyl-terminal peptide, CN-II, were purified from the CNBr digest of C5a des-Arg-74 by reverse-phase high performance liquid chromatography. The isolated derivatives were assessed for their abilities to cause contraction of isolated guinea pig ileal smooth muscle, guinea pig lung parenchymal strips, and degranulation of guinea pig polymorphonuclear neutrophils. In each assay, CN-I was devoid of biological activity at concentrations greater than 10(-6) M. In contrast, the carboxyl-terminal 56-residue fragment, CN-II, possessed weak (10(-6) versus 10(-9) M for classical anaphylatoxin) agonist activity in each of the assay systems. Our data suggest that structural information contained in the amino-terminal 17 residues of the C5a des-Arg-74 molecule contributes to the biological potency of the intact factor, but is not an essential component of the active site. Whether the structural information in residues 1-17 relates to receptor binding directly or serves to stabilize the conformation of the 18-73-fragment containing the active center of the molecule is yet to be determined.

Role of cyclooxygenase products in the lung action of leukotrienes A4, B4, C4, D4 and E4.

Leukotrienes (LT) LTA4, LTB4, LTC4, LTD4 and LTE4 induced marked contractions of guinea pig lung parenchymal strips mounted in organ baths. These contractions were inhibited differentially (40-50% for LTA4, LTC4, LTD4 and LTE4, and 90% for LTB4) by indomethacin (20 micrograms.ml-1; 55.9 microM). Two novel inhibitors of thromboxane synthetase (OKY-1581 and OKY-046) reduced the myotropic activity of the lung strips and the release of prostaglandins and thromboxanes from the perfused guinea pig lungs stimulated by LTB4 and LTD4. The release of cyclooxygenase products prostaglandin F2 alpha, thromboxane B2 and 12-hydroxyheptadecatrienoic acid by guinea pig lungs following stimulation with LTB4 and LTD4 was also measured by gas chromatography-mass spectrometry. The role of prostaglandins and thromboxanes in the lung actions of leukotrienes was confirmed using a cascade superfusion system and classical organ baths. Although prostaglandins and thromboxanes contribute to the contractile effect of LTB4 on the guinea pig lung whereas they may play a lesser role in the action of the peptidoleukotrienes (approx. 40-50%), stimulation of their release by the peptidoleukotrienes is many times more effective than by LTB4.

Interaction of various leukotrienes in guinea pig tracheal and lung parenchymal strips

Interaction of various leukotrienes in guinea pig tracheal and lung parenchymal strips

Spasmogenic activity of C5a desArg anaphylatoxin on guinea pig lung parenchymal strips: Sensitivity of the leukotriene-mediated component to cyclooxygenase inhibitors

The leukotriene-dependent component of C5a desArg-induced contractile activity on guinea pig lung parenchymal strips is inhibited by cyclooxygenase inhibitors. Indomethacin simultaneously increased leukotriene release while inhibiting both cyclooxygenase-dependent mediator release and the contractile force generated. Tissue responses to LTC 4 and LTD 4 are also inhibited by cyclooxygenase blockade, while contractions induced by the thromboxane A 2 analog, U-46619, histamine or acetylcholine are not affected. These data indicate a functional role for cyclooxygenase metabolites in leukotriene-induced contractile responses in lung.

Generation of leukotriene B 4 and leukotriene E 4 from porcine pulmonary artery

When chopped porcine pulmonary arteries were incubated with calcium ionophore A23187 (1) in the presence of indomethacin there was a time dependent generation of a substance which produced contractions of superfused strips of guinea-pig ileum smooth muscle (GPISM) which were indistinguishable from those induced by LTD 4. This material however had a different retention time from LTD 4 when subjected to HPLC and co-chromatographed with synthetic LTE 4. In addition to LTE 4 a substance which had properties indistinguisable from those of LTB 4 when assayed on a combination of guinea-pig lung parenchymal strips (GPP) and GPISM (2) was generated from the pulmonary artery. This substance co-chromatographed with synthetic LTB 4. The adventitia and intima were the richest source of LTE 4, the adventitia releasing slightly more than the intima. The output of LTB 4 and LTE 4 was inhibited by 6,9-deepoxy-6,9-(phenylimino)- Δ 6,8 prostaglandin I 1 (U-60,257). Nordihydroguaiaretic acid (NDGA) inhibited the generation of LTE 4.