Maan H. Saad

Bronchodilator‐mediated relaxation of normal and ovalbumin‐sensitized guinea‐pig airways: lack of correlation with lung adenylate cyclase activation

1 Isoprenaline, vasoactive intestinal peptide (VIP), prostaglandin E2 (PGE2) and forskolin caused a dose‐dependent relaxation of normal and ovalbumin‐sensitized guinea‐pig tracheal spirals and lung parenchymal strips in vitro. There was no difference in magnitude of relaxation or sensitivity to these relaxants between normal and sensitized tissues. The rank order of potency (concentration of each drug at which 50% of the maximum is obtained) for these relaxants on both trachea and parenchyma was VIP > isoprenaline > PGE2 > forskolin, although the parenchyma was more sensitive than the trachea. 2 The rank order of efficacy of the drugs used in relaxing both the trachea and lung parenchyma was isoprenaline (10 μm) > forskolin (30 μm) > VIP (0.1 μm) > PGE2 (10 μm). PGE2 at concentrations greater than 1 μm sometimes contracted the lung strip. 3 Pretreatment with indomethacin (8.5 μm), a cyclo‐oxygenase inhibitor, reduced the resting tone of tracheal spirals, but did not significantly affect the tone of lung strips. Indomethacin‐pretreatment did not affect drug‐induced relaxations of either normal or sensitized tracheal spirals. However, both normal and sensitized indomethacin‐pretreated lung strips relaxed significantly less (P<0.05) to isoprenaline, PGE2 and forskolin. Indomethacin‐pretreatment did not affect sensitivity of normal and sensitized trachea or parenchyma to the relaxant drugs. 4 All the relaxant drugs used stimulated adenylate cyclase activity in normal or sensitized lung parenchyma membrane preparations. The rank order of efficacy (maximal activation) was forskolin > isoprenaline = VIP > PGE2. There was no difference in response between normal and sensitized lungs. Adenylate cyclase activity of normal lung was stimulated as follows: forskolin (100 μm), 500.0 ± 50.0%; isoprenaline (100 μm), 186.0 ± 29.0%; VIP (10 μm), 213.0 ± 19.0% and PGE2 (100 μm), 155.0 ± 23.0% of basal activity. Similar values were obtained for sensitized lung parenchyma. 5 Indomethacin pretreatment did not significantly affect normal or sensitized lung adenylate cyclase stimulation by isoprenaline, VIP, forskolin or PGE2. 6 It was concluded that: (a) Immunological sensitization to ovalbumin does not induce hypoactivity of relaxant drug receptors and/or the adenylate cyclase system of the airway tissues of the guinea‐pig. (b) There is an apparent lack of correlation between tissue relaxation in vitro and adenylate cyclase activity since the rank order of the efficacy of a range of relaxants was different for the two effects and furthermore indomethacin‐treatment of airway tissues yielded differential results.

Release of leukotriene C4 from guinea pig trachea

Immunological (ovalbumin) and non-immunological (calcium ionophore A23187) stimulation of guinea pig trachea induces a prolonged contraction that is enhanced by indomethacin (8.5 microM) and inhibited by nordihydroguaiaretic acid (50 microM) pretreatment of the tissue. The mediator released by the above stimuli was identified as leukotriene C4 by reverse-phase high performance liquid chromatography, and quantitated by bioassay. Indomethacin, and/or arachidonic acid (32.8 microM) did not enhance the release, whereas nordihydroguaiaretic acid reduced the contraction and release of LTC4. The results demonstrate the hitherto unproved capability of the large airways to synthesize leukotrienes and emphasize the importance of examining their role in asthma.

Mediators of arachidonic acid‐induced contractions of indomethacin‐treated guinea‐pig airways: leukotrienes C4 and D4

1 Arachidonic acid (AA) (66 μm) induced contractions of indomethacin‐treated (8.4 μm) guinea‐pig tracheal and lung parenchymal preparations. Mepacrine (210 μm) treatment did not affect the magnitude of contraction induced by AA. 2 Normal and ovalbumin‐sensitized tissues responded identically to AA, and released equivalent amounts of the contractile mediators. 3 Nordihydroguaiaretic acid (100 μm) markedly reduced release of the contractile mediators and reduced AA‐induced contractions of the airways. 4 The mediators of AA‐induced, calcium ionophore A23187‐induced, and antigen‐induced contraction of the trachea and lung parenchyma were purified and identified by reverse‐phase high performance liquid chromatography to be leukotrienes C4 and D4, being present in an approximate ratio of 20:1. 5 Mepacrine‐treated trachea exhibited a smaller contractile response to stimuli (A23187 for normal tissues and ovalbumin for sensitized tissues). Addition of exogenous AA (66 μm) increased the magnitude of contraction, although not to the level observed on tissues not treated with mepacrine. There was no observable effect of AA on the response of mepacrine‐treated parenchyma to the ionophore or antigen. 6 It was concluded that (a) immunological sensitization does not alter AA metabolism via the lipoxygenase pathway in guinea‐pig airways and (b) the mediators of AA‐induced contraction are leukotrienes C4 and D4.

Specific binding of leukotriene B4 to guinea pig lung membranes

We have demonstrated binding sites for LTB4 in guinea pig lung membranes. Binding of [3H]-LTB4 was of high affinity (Kd = 0.76 nM), saturable and linear with protein concentration (0.2-1.2 mg/ml). Scatchard and Hills plot analysis indicated a single class of binding site with a Hills coefficient of 0.99 +/- 0.08 (n = 4). [3H]-LTB4 was unmetabolized during incubation with membrane preparations, as indicated by high performance liquid chromatography. Divalent cations such as Mg2+ and Ca2+ enhanced binding capacity without changing the Kd. Na+ ions decreased binding in a concentration-dependent manner. Guanine nucleotides, GTP, GTP gamma S and Gpp(NH)p also decreased the number of binding sites. Finally, competition experiments demonstrated the following order of potency for displacement of [3H]-LTB4 from its receptor site: LTB4 greater than 20-OH-LTB4 much greater than 20-COOH-LTB4 = 6-trans-12-epi-LTB4 greater than LTC4 = LTD4 = 5-HETE. These data indicate that a specific LTB4 receptor, in addition to the previously documented LTC4 and LTD4 receptors, exists in guinea pig lung.

Role of calcium in arachidonic acid-induced contractions of guinea pig airways

We have previously shown that arachidonic acid (AA)-induced contractions of indomethacin-pretreated guinea pig trachea and parenchyma are due to the synthesis of leukotrienes C4 and D4. The present experiments were designed to investigate the role of calcium (Ca2+) in the above. AA (66 microM)-induced contractions of trachea, but not parenchyma, were reduced in Ca2+-free Krebs-Henseleit solution ( KHS ). However the contractions of both trachea and parenchyma were abolished in Ca2+-free KHS with either lanthanum chloride (1 mM) or EDTA (300 microM). The Ca2+ antagonists, verapamil (100 microM), nitrendipine (100 microM), and TMB-8 (100 microM), reduced AA-induced contractions of both trachea and parenchyma. Re-addition of Ca2+ (2.2 mM) to trachea and parenchyma in Ca2+-free KHS in the presence of lanthanum restored the AA-induced contractions. This effect of Ca2+ was reduced by verapamil (100 microM) or nitrendipine (100 microM). LTC4-induced contractions of trachea and parenchyma were unaffected by nitrendipine (100 microM), whereas tracheal contractions were reduced in Ca2+-free KHS . Both tracheal and parenchymal contractions to LTC4 were reduced in Ca2+-free KHS in the presence of lanthanum chloride (1 mM). We conclude that superficially bound pools of Ca2+ are important in AA-induced contractions of the airways. Furthermore, nitrendipine reduces AA-induced contractions by inhibiting AA metabolism and not by inhibiting airway smooth muscle contraction induced by released leukotrienes.

Isolation of leukotriene C4 from human seminal fluid

LTC4 was isolated and characterized from seminal fluid of seven human volunteers. A compound with a similar retention time to that of synthetic LTC4 was obtained using reverse-phase high performance liquid chromatography. The ultraviolet absorbance of the extracted substance was identical to synthetic LTC4. Furthermore this compound contracted the guinea pig ileum and lung parenchymal strip. Its effects were antagonized by the leukotriene antagonist FPL55712. It was concluded that LTC4 is present in human seminal fluid in very small amounts (about 100 ng/ejaculate). The possible physiological functions of LTC4 in the reproductive tract are discussed.

Dual regulation of neutrophil adenylate cyclase by fluoride and its relationship to cellular activation

1 Fluoride stimulated (1–10 mm) and inhibited (10–100 mm) adenylate cyclase of neutrophil membranes in a GTP‐independent manner. The latter fluoride concentration range corresponded to that shown previously to induce cellular responses. 2 Dual regulation of cyclase activity was also exhibited by a nonhydrolysable GTP analogue, guanosine 5′[γ‐thio]triphosphate (GTPγS). Inhibition was observed at 0.1–10 nm GTPγS while stimulation occurred at > 10 nM GTPγS. 3 Relatively high levels (> μm) of formylmethionyl‐leucyl‐phenylalanine inhibited adenylate cyclase in the presence of GTP (10 μm). 4 Pertussis toxin pretreatment abolished adenylate cyclase inhibition mediated by GTPγS and formylmethionyl‐leucyl‐phenylalanine but did not influence fluoride‐induced inhibition.