Mechanism of A23187-induced airway obstruction in the guinea pig

Exposure of conscious guinea pigs to A23187 aerosol produced a concentration-related increase of excised lung gas volume (ELGV), . ., postmortem pulmonary gas trapping. Measurements of ELGV were highly correlated with measurements of dynamic compliance (C_dyn) and total pulmonary resistance (R_L) and were used as an indication of airway obstruction. We pretreated guinea pigs intravenously with the following drugs: atropine; LY163443, a selective LTD₄/E₄ antagonist; indomethacin; propranolol; and pyrilamine. The guinea pigs were exposed for 8 minutes to the A23187 aerosol, and ELGV measurements were then made. Atropine or pyrilamine prevented the A23187-induced gas trapping. Indomethacin or propranolol tended to potentiate the response and when combined, they potentiated the gas trapping by 80%. LY163443 had no effect alone, but when combined with indomethacin, propranolol, and pyrilamine, inhibited A23187-induced gas trapping by 67%. We conclude that cholinergic and histaminergic mechanisms play major roles in the ionophore-induced pulmonary gas trapping of the guinea pig. With appropriate pretreatment, sulfidopeptide leukotrienes may produce a substantial effect.     

Propranolol potentiates leukotriene D4-induced bronchoconstriction and enhances antagonism by FPL 55712

Aerosol LTD4-induced bronchoconstriction in anesthetized, spontaneously breathing guinea pigs was potentiated by either pretreatment with propranolol or bilateral adrenalectomy, whereas bilateral vagotomy did not affect the LTD4 response. The dose-response curve describing LTD4-induced changes in dynamic lung compliance (CDYN) and pulmonary resistance (RL) [as reflective indices of bronchoconstriction] was shifted to the left by approximately 20-fold by propranolol. Against an equal degree of LTD4-induced bronchoconstriction, the leukotriene antagonist, FPL 55712, had an apparent 20-fold greater potency in propranolol-pretreated animals vis a vis saline-treated controls. The duration of action of aerosol FPL 55712 was similar in both propranolol-treated and saline-treated animals. These results demonstrate that aerosol LTD4-induced bronchoconstriction is modulated by an adrenergic compensatory bronchodilator mechanism that is apparently dependent upon the adrenals and independent of vagal influences. Inhibition of the effect of this reflex with propranolol also enhances the apparent potency of an aerosol leukotriene antagonist, FPL 55712, presumably reflecting a constant LTD4 to antagonist ratio in the saline-treated and propranolol-pretreated guinea pigs.     

Leukotriene D4 and platelet-activating factor-acether antagonists on allergic and arachidonic acid-induced reactions in guinea pig airways

Arachidonic acid (AA) and ovalbumin (OA) were used to induce contractions of sensitized guinea pig tracheal spiral (indomethacin-pretreated) and lung parenchymal strip preparations. This model was used to examine the properties of three leukotriene (LT) D4 antagonists and a platelet-activating factor (PAF)-acether receptor antagonist. The three LTD4 antagonists, L-649,923, FPL 57231, and LY163443, inhibited AA-induced contractions of indomethacin-pretreated tracheal spirals selectively. The PAF-acether antagonist, L-652,731, did not inhibit AA-induced contractions of either trachea or parenchyma. This confirmed that AA-induced contractions of trachea involved release and activity of LTD4. The LTD4 antagonists and L-652,731 partially inhibited OA-induced contractions of both trachea and parenchyma. When L-649,923 and L-652,731 or FPL 57231 and L-652,731 were combined, an additive inhibitory effect on OA-induced contractions was observed. When LY163443 and L-652,731 were combined, the inhibitory effect was synergistic. This may be due to the additional effect of LY163443 to inhibit phosphodiesterase. Total inhibition of OA-induced contractions was obtainable with relatively low concentrations when a LTD4 and PAF-acether antagonist were combined. These results suggested that LTD4 and PAF-acether may be the two major mediators in our model of allergic bronchospasm. The LTD4 and PAF-acether antagonists had the capacity to decrease baseline tone, even on tissues that were already relaxed with indomethacin, suggesting that LTD4 and PAF-acether may contribute to intrinsic tone in airway smooth muscle.     

Propranolol potentiates leukotriene D4-induced bronchoconstriction and enhances antagonism by FPL 55712

Aerosol LTD₄-induced bronchoconstriction in anesthetized, spontaneously breathing guinea pigs was potentiated by either pretreatment with propranolol or bilateral adrenalectomy, whereas bilateral vagotomy did not affect the LTD₄ response. The dose-response curve describing LTD₄-induced changes in dynamic lung compliance (C_{D Y N}) and pulmonary resistance (R_L) [as reflective indices of bronchochonstriction] was shifted to the left by approximately 20-fold by propranolol. Against an equal degree of LTD₄-induced bronchoconstriction, the leukotriene antagonist, FPL 55712, had an apparent 20-fold greater potency in propranolol-pretreated animals vis a vis saline-treated controls. The duration of action of aerosol FPL 55712 was similar in both propranolol-treated and saline-treated animals. These results demonstrate that aerosol LTD₄-induced bronchoconstriction is modulated by an adrenergic compensatory bronchodilator mechanism that is apparently dependent upon the adrenals and independent of vagal influences. Inhibition of the effect of this reflex with propranolol also enhances the apparent potency of an aerosol leukotriene antagonist, FPL 55712, presumably reflecting a constant LTD₄ to antagonist ratio in the saline-treated and propranolol-pretreated guinea pigs.     

Bronchoconstrictor effects of leukotriene B4 in the guinea pig in vivo

Administration of leukotriene B4 (LTB4) to anesthetized spontaneously breathing guinea pigs either by the intravenous or aerosol route produced pronounced changes in pulmonary resistance and dynamic compliance. The effects were short lived and were completely abolished by pretreatment of animals with the cyclooxygenase inhibitor indomethacin. Histological examination of lungs following aerosol administration of LTB4 showed a pronounced neutrophil infiltration. These results confirm previous in vitro studies in which LTB4 was shown to produce contractions on guinea pig parenchymal strips indirectly by releasing thromboxane A2.     

Bronchoconstrictor effects of leukotriene B4 in the guinea pig

Administration of leukotriene B₄ (LTB₄) to anesthetized spontaneously breathing guinea pigs either by the intravenous or aerosol route produced pronounced changes in pulmonary resistance and dynamic compliance. The effects were short lived and were completely abolished by pretreatment of animals with the cyclooxygenase inhibitor indomethacin. Histological examination of lungs following aerosol administration of LTB₄ showed a pronounced neutrophil infiltration. These results confirm previous studies in which LTB₄ was shown to produce contractions on guinea pig parenchymal strips indirectly by releasing thromboxane A₂.     

Effects of propranolol and indomethacin on muscarinic airway reactivity in unanesthetized guinea pigs

To investigate whether endogenous beta-adrenergic stimulation or cyclooxygenase products normally affect muscarinic reactivity in conscious, spontaneously breathing guinea pigs, we measured specific airway resistance (SRaw) during acetylcholine (ACh) infusion before and after treatment with propranolol (10 mg/kg ip) or indomethacin (30 mg/kg ip). Airway reactivity was assessed by measuring changes in SRaw upon increasing ACh infusion. We found that propranolol treatment increased reactivity to parenteral ACh, but did not change baseline SRaw. Furthermore, propranolol reduced the range in muscarinic reactivity for the group, and it enhanced thr reproducibility of measurements in individual animals. In contrast, indomethacin had no effect on either baseline SRaw or muscarinic reactivity. Our results suggest that beta-blockade of endogenous adrenergic stimulation increases the muscarinic reactivity of guinea pig airways, but does not influence resting airway tone. It appears that propranolol treatment allows a more reproducible assessment of muscarinic reactivity in the guinea pig. In contrast, cyclooxygenase products do not seem to significantly affect baseline airway resistance, reactivity, or reproducibility in the guinea pig.     

Bronchoconstrictor effects of leukotriene B4 in the guinea pig in vivo

Administration of leukotriene B₄ (LTB₄) to anesthetized spontaneously breathing guinea pigs either by the intravenous or aerosol route produced pronounced changes in pulmonary resistance and dynamic compliance. The effects were short lived and were completely abolished by pretreatment of animals with the cyclooxygenase inhibitor indomethacin. Histological examination of lungs following aerosol administration of LTB₄ showed a pronounced neutrophil infiltration. These results confirm previous $\text{in vitro}$ studies in which LTB₄ was shown to produce contractions on guinea pig parenchymal strips indirectly by releasing thromboxane A₂.     

Contribution of lipoxygenase and cyclooxygenase metabolites in the modulation of cyclic nucleotides in the guinea pig myometrium. Differential effects of carbachol and ionophore A23187

Accumulation of cyclic GMP in estradiol-treated immature guinea pig myometrium was enhanced by carbachol, ionophore A23187, unsaturated fatty acids and their hydroperoxides. Cyclic AMP content was elevated only by arachidonic acid, A23187 and PGI2. Eicosatetraynoic acid (TYA), but not indomethacin prevented all cyclic GMP responses. The effects of A23187 and arachidonate on cyclic AMP were accompanied by a parallel increase (2-3 fold) in the generation of PGI2 by the myometrium. Both events were similarly reduced by indomethacin, TYA, 15-hydroperoxyarachidonic acid and tranylcypromine, suggesting that PGI2 was involved. Omission of Ca2+ or addition of mepacrine or p-bromophenacylbromide abolished the stimulatory effects of A23187 and carbachol on cyclic GMP as well as the A23187-induced elevations in both PGI2 and cyclic AMP generation. Thus, with both exogenous arachidonate as well as with endogenous fatty acid, released through an apparent phospholipase A2-induced activation process, the lipoxygenase pathway was associated with an activation of the cyclic GMP system and the cyclooxygenase pathway, via PGI2 generation, with an activation of the cyclic AMP system. Carbachol failed to alter both cyclic AMP content and the release of PGI2 suggesting a cholinergic receptor-mediated fatty acid release process, selectively coupled to the lipoxygenase route.     

Pharmacological model for airway hypersensitivity produced by propranolol and reserpine in guinea pigs

Combined treatment with propranolol and reserpine enhanced acetylcholine-induced doseresponse curves for bronchoconstriction in guinea pigs in vivo. This airway hyperreactivity model was investigated pharmacologically. (1) Increased capillary permeability and increases in leukocytes in bronchoalveolar lavage fluid (BALF) were not observed after this combined treatment. (2) The increased airway sensitivity to acetylcholine produced by propranolol and reserpine was inhibited by ketotifen and theophylline, reported in clinical studies to inhibit airway hyperreactivity. (3) Two leukotriene (LT) receptor antagonists, MCI-826 and FPL-55712, clearly inhibited this increased airway reactivity. (4) A thromboxane A₂ (TXA₂) receptor antagonist, ONO-3708, and TXA₂ synthetase inhibitor, OKY-046, also inhibited this increased airway reactivity.These results suggest that the airway hyperreactivity model produced by propranolol and reserpine in guinea pigs is a valuable pharmacological tool for investigating a remedy and LT and TXA₂ may be involved in the onset of this airway hyperreactivity.     

Prevention of abnormal pulmonary mechanics in the postmortem guinea pig lung

Severe postmortem bronchoconstriction has been shown previously in guinea pig lungs and linked to pulmonary blood loss during exsanguination (Lai et al., J. Appl. Physiol. 56: 308-314, 1984). To reexamine this phenomenon we measured postmortem airway function in anesthetized open-chest guinea pigs after sudden circulatory arrest. Animals were divided into 4 groups of 10 and ventilated for 15 min postmortem with different gases: 1) room air, 2) conditioned air, 3) dry 5% CO2-21% O2-74% N2, and 4) conditioned 5% CO2-21% O2-74% N2. In room air-ventilated lungs there was a 50% decrease in dynamic compliance (Cdyn) by 15 min and marked gas trapping compared with control lungs. Conditioning the room air did not attenuate these changes, but when 5% CO2 was added to the conditioned postmortem inspirate, gas trapping was eliminated and the fall in Cdyn was almost abolished. Ventilation with a dry 5% CO2 gas mixture at room temperature resulted in a 31% fall in Cdyn at 15 min but no gas trapping. We conclude that marked abnormalities of airway function occur postmortem in room air-ventilated guinea pig lungs in the absence of pulmonary blood loss. The changes are mainly due to airway hypocarbia, a known cause of bronchoconstriction, but a reduction in Cdyn can also occur if there is marked airway cooling and drying. Acute postmortem airway dysfunction can be prevented in the guinea pig by maintaining normal airway gas composition.     

Experimental edema and the activity of the pulmonary surfactant system during the suppression of prostaglandin synthesis

Experiments on 23 white rats and 10 guinea pigs have shown that preliminarily indomethacin-induced inhibition of prostaglandins synthesis prevented development of pulmonary oedema, evoked by heterologous serum in rats and by vagotomy in guinea pigs. Fourfold infusion of indomethacin in experiments on 29 rats decreased extracellular fraction activity of the pulmonary surfactant and exhausted its cellular reserve.     

Effect of BI-L-239, A-64077 and MK-886 on leukotriene B4 synthesis by chopped guinea pig lung and on antigen-induced tracheal contraction in vitro.

The 5-lipoxygenase (5-LO) inhibitors BI-L-239 and A-64077 were compared with the 5-LO translocation inhibitor MK-886 for the ability to inhibit leukotriene B4 (LTB4) biosynthesis by chopped (1 mm3) guinea pig lung. LTB4 synthesis by ovalbumin-sensitized chopped lung tissue was determined after stimulation with either calcium ionophore (A23187) or antigen. With A23187 stimulation, MK-886 was more potent (IC50 = 0.39 +/- 0.23 microM, mean +/- SEM, p < 0.01) than BI-L-239 (IC50 = 2.48 +/- 0.46 microM) or A-64077 (IC50 = 4.68 +/- 0.70 microM) and BI-L-239 was more potent than A64077 (p < 0.02). Thus, the order of potency was MK-886 > BI-L-239 > A-64077 for inhibition of calcium ionophore-induced LTB4 generation. There was no significant differences in potency of the compounds in chopped lung stimulated with antigen: IC50 for LTB4 synthesis by A-64077 = 3.31 +/- 1.70 microM, for BI-L-239 = 9.06 +/- 4.94 microM, and for MK-886 = 13.33 +/- 7.91 microM. The ability of these compounds to inhibit contraction of tracheal tissue from actively sensitized guinea pigs in response to antigen was also determined in the presence of indomethacin (15 micrograms/ml), mepyramine, and atropine (5 micrograms each/ml). Both 5-LO inhibitors inhibited antigen-induced contraction, with IC50 values for BI-L-239 and A-64077 of 1.58 and 4.35 microM respectively. MK-886 was ineffective at inhibiting antigen-induced tracheal contraction in vitro at concentrations up to 30 microM. In summary, these compounds inhibit antigen-induced and A23187-induced leukotriene biosynthesis in guinea pig tissue. These 5-LO inhibitors were similarly effective at inhibiting antigen-induced tracheal contraction where MK-886 was ineffective.     

Differing mechanisms for leukotriene D4-induced bronchoconstriction in guinea pigs following intravenous and aerosol administration

Leukotriene D₄ (LTD₄) when administered intravenously or by aerosol to guinea pigs produced changes in pulmonary mechanics including a decrease in dynamic compliance and an increase in pulmonary resistance. The effects of intravenous LTD₄ (0.5 μg kg⁻¹) were short lived and abolished by pretreatment of the animal with either cyclooxygenase inhibitors, a thromboxane synthetase inhibitor (OKY 1555) or an SRS-A antagonist (FPL 55712). These findings suggest that bronchoconstriction produced by the intravenous infusion of LTD₄ at 0.5 μg kg⁻¹ is due to the release of thromboxane A₂. However, in animals treated with indomethacin, LTD₄ at higher doses (>0.8 μg kg⁻¹) still elicited a bronchoconstriction which could be blocked by FPL 557112. Nebulization of 0.1 – 1.0 μg of LTD₄ into the lung produced prolonged changes in pulmonary mechanics which were inhibited by FPL 55712 and were potentiated indomethacin. LTD₄, therefore, when administered by aerosol produced effects on the lung which were not mediated by cyclooxygenase products. Responses to nebulized rather than intravenous LTD₄ in the guinea pig may more closely resemble those seen in human tissues.     

Effects of obsidan on the development of pulmonary edema and hemodynamics of the lesser circulation after administration of noradrenaline

In experiments on white rats, guinea pigs and cats it was shown that preliminary infusion of propranolol sharply increases the edemagenous lungs sensitiveness to the infusion of exogenous norepinephrine of white rats and guinea pigs. The infusion of the propranolol to cats leads to a decrease of volume blood flow velocity in the pulmonary artery with the simultaneous difficulty of the outflow in pulmonary veins and to increase of hydrostatic pressure in the lesser circulation. The infusion on that background of epinephrine called the development of temporary sharp hearts failure with the sharp increase of the pressure in pulmonary artery (to 196%) and systemic pressure.     

Repeated antigen challenge induces airway hyperresponsiveness with tissue eosinophilia in guinea pigs

To test the hypothesis that the development of airway hyperresponsiveness (AHR) lasting greater than or equal to 3 days after the last antigenic exposure required repeated mediator release, we compared dose-response changes in lung resistance (RL) to acetylcholine (ACh) in animals sensitized with 1% ovalbumin (OA), 4% Bordatella pertussis aerosol and subsequently challenged with 0.5% OA aerosol twice weekly for 4-6 wk vs. animals receiving saline aerosol instead of OA. Despite antihistamine pretreatment, each OA challenge produced cyanosis and inspiratory indrawing. Blood gas analysis in six guinea pigs revealed an immediate fall in arterial PO2 (PaO2) from 104.3 +/- 4.9 to 35.4 +/- 2.2 Torr after a 1-min exposure to aerosolized OA. ACh dose-response measurements of RL 3 days after the last OA challenge demonstrated a leftward shift and an increased magnitude of response. These differences were less marked at 7 days, and by 14 days after the last OA challenge, ACh dose-response curves were not different from those of control guinea pigs. Sensitization without repeated antigen challenge did not cause hyperresponsiveness. Morphometric analysis showed significantly increased numbers of eosinophils in the epithelium of airways in hyperresponsive guinea pigs, without neutrophil infiltration or alterations in epithelium and airway wall areas. We conclude that repeated antigenic challenge, but not sensitization alone, causes prolonged AHR in guinea pigs, which is associated with tissue eosinophilia.     

Inhibitory effect of valves on endothelium-dependent relaxations to calcium ionophore in canine saphenous vein

The present study was designed to evaluate endothelium-dependent relaxation to the calcium ionophore A-23187 in isolated canine saphenous veins. Isometric force recordings and cGMP measurements using isolated veins with and without valves were performed. During contractions to U-46619 (3 x 10(-7) M), endothelium-dependent relaxations to A-23187 (10(-9)-10(-6) M) were significantly reduced in rings with valves compared with rings without valves. Endothelial removal abolished A-23187-induced relaxation. Relaxations to forskolin (FK; 10(-8)-10(-5) M) and diethylaminodiazen-1-ium-1,2-dionate; DEA-NONOate, 10(-9)-10(-5) M) were identical in rings with and without valves. In rings without valves, a nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME; 3 x 10(-4) M), and a cyclooxygenase inhibitor, indomethacin (10(-5) M), partially reduced A-23187-induced relaxation. However, in rings with valves, L-NAME had no effect, whereas indomethacin abolished the relaxation to A-23187. A selective soluble guanylate cyclase inhibitor, 1H-[1,2,4]-oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 3x10(-6) M), had no effect on the relaxation to A-23187 in either group. In contrast, ODQ abolished the A-23187-induced increase in cGMP levels, suggesting that relaxation to nitric oxide released by A-23187 is independent of increases in cGMP. These results demonstrate that endothelium-dependent relaxation to A-23187 is reduced in regions of veins with valves compared with relaxation in the nonvalvular venous wall. Lower production of nitric oxide in endothelial cells of valvular segments appears to be a mechanism responsible for reduced reactivity to A-23187.     

Ionophore-induced disassembly of blood platelet microtubules: effect of cyclic AMP and indomethacin

Cytoplasmic calcium levels are believed to be important in blood platelet activation. Upon activation, the discrete marginal microtubule band, which maintains the discoid shape of non-activated platelets, becomes disrupted. Present studies demonstrate that the extent of assembly of the marginal microtubule band is related to cytoplasmic calcium levels. The divalent cationophore, A23187, causes platelet aggregation, secretion, and contraction by promoting calcium transport from intraplatelet storage sites into the cytoplasm. A23187 caused disassembly of platelet microtubules. Quantitation of electron micrographs revealed that numbers of microtubules were reduced by approximately 80% after A23187 treatment. Secondly, assembled microtubules in homogenates of platelets, in which microtubules were stabilized prior to homogenization, were decreased in favor of free tubulin in A23187-treated platelets. Thirdly, A23187 increased 14C-colchicine binding by intact platelets; this also indicated a shift in the microtubule subunit equilibrium to favor free, colchicine-binding tubulin subunits. In control experiments, A23187 did not affect the stability of platelet tubulin, the colchicine binding reaction, or the total tubulin content of platelets. Stimulation of colchicine binding depended on A23187 concentration (0.05-0.5 microM) and did not require extracellular calcium. A23187-stimulation of colchicine binding was blocked by dibutyryl cyclic AMP (0.80 mM) and/or 3-isobutyl-1-methylxanthine (50 microM) and by indomethacin (10 microM). Cyclic AMP or indomethacin also interferes with A23187-induced platelet activation, but indomethacin is not likely to completely inhibit the perturbation of intraplatelet calcium gradients by A23187. It is suggested that A23187-induced microtubule disassembly may be an indirect effect of calcium on microtubules.     

Increased in vitro responses of tracheal smooth muscle from hyperresponsive guinea pigs

Repeated aerosol antigen challenge of previously sensitized guinea pigs induces airway hyperresponsiveness to inhaled acetylcholine. To determine the mechanism producing these airway changes and assuming that changes in the trachealis muscle reflect changes in muscle of the entire tracheobronchial tree, we examined the in vitro smooth muscle mechanics and morphometric parameters of tracheae from guinea pigs demonstrating hyperresponsiveness in vivo vs. tracheae from control guinea pigs. No differences between these groups were found in luminal volume at zero transmural pressure, passive pressure-volume characteristics, or area of airway wall. Smooth muscle areas were slightly less in tracheae from hyperresponsive guinea pigs. Tracheae from hyperresponsive guinea pigs had both significantly increased isovolumetric force generation and isobaric shortening compared with tracheae from controls when evaluated over the range of transmural pressures from -40 to 40 cmH2O. We conclude that the in vivo airway hyperresponsiveness induced with repeated antigen challenge is associated with both increased force generation and shortening of tracheal smooth muscle without increased muscle mass, suggesting enhanced contractile activity.     

Tobacco smoke exposure and bombesin-like peptides in guinea pigs

Bombesin-like peptides (BLPs) are associated with tobacco smoke (TS)-induced diseases. We sought to determine if acute TS exposure releases BLPs into the pulmonary circulation. Sensitized and non-sensitized guinea pigs were chronically exposed to TS or compressed air. Thereafter, the lungs were acutely challenged with TS while perfused. Perfusates were analyzed for BLPs. TS increased BLPs in non-sensitized guinea pigs. A separate study determined daily bombesin exposure increased lung cell counts but not airway hyperresponsivensess. TS exposure releases BLPs into the pulmonary circulation but can be modified by host factors and bombesin itself does not induce airway hyperresponsiveness.