John F. Burka

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.

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.

Effects of calcium channel blockers and a calmodulin antagonist on contractions of guinea pig airways

Both antigen- and calcium ionophore A23187-induced airways contractions are dependent on increased concentrations of free intracellular calcium. Antigen, but not A23187, contracted airways in the absence of extracellular calcium, suggesting that antigen-induced airways contraction was partly dependent on the mobilization of intracellular calcium. A series of calcium entry blockers and a calmodulin antagonist were examined on airways contraction induced by antigen, A23187, histamine, and LTD4. Verapamil (10(-5)-3 X 10(-4) M) and nitrendipine (3 X 10(-5)-2.8 X 10(-4) M) dose-dependently inhibited antigen-induced contraction of trachea but only partly inhibited antigen-induced contraction of parenchyma and A23187-induced contractions of both trachea and parenchyma. Lanthanum chloride (up to 10(-4) M) and flunarizine (up to 2.1 X 10(-5) M) were relative ineffective. High concentrations of trifluoperazine (1-3 X 10(-4) M) were necessary to inhibit antigen- and A23187-induced airways contractions. Only verapamil and trifluoperazine were effective inhibitors of the responses to histamine and LTD4. In light of the relative ineffectiveness of the above calcium antagonists on the LTD4 dose-response curves, it is suggested that airways contraction is dependent on mobilization of intracellular calcium and that the action of nitrendipine and verapamil as inhibitors of antigen- and A23187-induced contractions is at the point of blocking mediator (i.e. LTC4 and LTD4) release. Verapamil and trifluoperazine also appear to affect contractions. However, the high concentrations of calcium channel and calmodulin antagonists to inhibit antigen- and A23187-induced contractions may limit their usefulness as anti-allergic, anti-asthmatic agents.

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.

Pharmacological modulation of responses of guinea‐pig airways contracted with antigen and calcium ionophore A23187

1 Ovalbumin (OA) and calcium ionophore A23187 were used to induce contractions of sensitized guinea‐pig tracheal and lung parenchymal preparations in the presence and absence of indomethacin. This model was used to examine the properties of a series of compounds reported to inhibit 5‐lipoxygenase or to antagonize lipoxygenase products at the receptor level. 2 FPL55712 and piriprost appeared to act as pharmacological antagonists because they rapidly reduced tracheal tone established by OA. 3 The prolonged phase (i.e. > 10 min post‐challenge) of airways contractions induced by OA is assumed to be lipoxygenase‐dependent and was inhibited by nordihydroguaiaretic acid (NDGA), FPL55712, nafazatrom and benoxaprofen, in order of potency. Piriprost had similar inhibitory effects on the trachea, but not on lung parenchyma. The inhibitory effects of NDGA and FPL55712 were reduced, and those of nafazatrom increased by indomethacin. Indomethacin decreased the inhibitory effect of piriprost on the trachea, but facilitated inhibition by this agent on the parenchyma. A roughly similar pattern was seen on A23187‐induced contractions, but FPL55712 did not modify these contractions and benoxaprofen enhanced the response of the trachea. 4 BW755C enhanced the overall contractile response of OA‐ and A23187‐induced tracheal contractions (but not parenchymal contractions) in a bell‐shaped manner, an effect not seen in the presence of indomethacin. This indicated that BW755C could be acting as a cyclo‐oxygenase inhibitor. 5 The results suggested that, although inhibitors of the lipoxygenase pathway were partially effective in inhibiting the contractions of the airways induced by OA or A23187, other mediators in addition to those of the lipoxygenase pathway contribute to these contractions.

Pharmacological modulation of responses of guinea-pig airways contracted with arachidonic acid

1 Arachidonic acid (AA) was used to induce contractions of guinea‐pig tracheal and lung parenchymal preparations in the presence of indomethacin. 2 Prior addition of FPL55712, nordihydroguaiaretic acid (NDGA), piriprost, benoxaprofen or nafazatrom, in order of potency, inhibited AA‐induced contractions of trachea. Higher concentrations (2–3 fold) were necessary to inhibit contractions of parenchyma. 3 FPL55712 and piriprost appeared to act as pharmacological antagonists of leukotrienes because they rapidly reduced the tone of the airways established by AA. 4 Administration of exogenous AA to indomethacin‐treated trachea appears to be a good model to examine leukotriene receptor antagonists and inhibitors of the lipoxygenase pathway.

ANTIGEN‐INDUCED CONTRACTION OF GUINEA‐PIG TRACHEA: SEARCH FOR MEDIATOR RELEASE WITH CASCADE SUPERFUSION BIOASSAY

Ovalbumin induces contraction of sensitized guinea‐pig trachea, both in the presence and absence of indomethacin. Cascade superfusion bioassay was employed to detect mediator release. A prostaglandin E‐like material was released in response to antigen in the absence of indomethacin but, no biologically active material could be detected from trachea in the presence of indomethacin. Thus the substance responsible for antigen‐induced tracheal contraction, although appearing to be a lipoxygenase product, is as yet undefined.

Studies on the role of arachidonic acid metabolites in airways contraction induced in vitro by antigen and calcium ionophore A23187.

The effects of inhibitors of the cyclooxygenase and lipoxygenase pathways of arachidonic acid (AA) metabolism were studied on the contractions of guinea pig tracheal spirals and lung parenchymal strips induced by antigen and calcium ionophore A23187. Inhibition of the cyclooxygenase pathway with indomethacin results in enhancement of antigen- and A23187-induced contraction of trachea which is a result of diversion of AA into the lipoxygenase pathway and inhibition of modulatory cyclooxygenase products. Indomethacin does not enhance parenchymal contractions but partly inhibits contraction induced by a strong stimulus (5.7 μM A23187 or 100 μg/ml ovalbumin). Parenchymal contraction is inhibited by agents that block the lipoxygenase pathway of AA metabolism, phenidone and nordihydroguaiaretic acid. These agents inhibit the prolonged phase of antigen-induced tracheal contraction but in some cases enhance the early phase of tracheal contraction induced by antigen or A23187. The results suggest that contraction of parenchyma induced by antigen or A23187 are the result of a bronchoconstrictor lipoxygenase product and only have a cyclooxygenase bronchoconstrictor component following a strong stimulus. In contrast, contraction of trachea also appears to be the result of a bronchoconstrictor lipoxygenase product which may not be identical to that contracting parenchyma, and is modulated by cyclooxygenase products. The results emphasize the importance of comparing small and large airways to further our understanding of asthmatic bronchoconstriction.

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.