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1. In the present study, we have investigated the role of kinins in allergen-induced bronchoconstriction. 2. Anaesthetized guinea-pigs were sensitized to ovalbumin, ventilated artificially, pretreated with atropine (1.4 mumol kg-1, i.v.) and total pulmonary resistance (RL) measured. In preliminary studies in the presence of the neutral endopeptidase inhibitor, phosphoramidon (4.5 mumol kg-1, i.v.), the bradykinin B2 receptor antagonist Hoe 140 (0.1 mumol kg-1, i.v.) completely abolished the increase in RL following aerosolized bradykinin (1 mM, 40 breaths), but had no effect on the increase in RL following aerosolized neurokinin A (NKA, 10 microM, 40 breaths). On the other hand, a combination of the NK1 (CP-96,345, 2 mumol kg-1, i.v.) and NK2 (SR 48968, 0.3 mumol kg-1, i.v.) tachykinin receptor antagonists abolished completely the increase in RL produced by NKA and partially inhibited the increase in RL produced by bradykinin. These results confirm previous studies that suggest that bradykinin induces the release of tachykinins from sensory nerves in guinea-pig airways. 3. Aerosolized ovalbumin (0.5%, 5 breaths) increased RL in sensitized guinea-pigs pretreated with atropine (1.4 mmol kg-1, i.v.), an effect that began within 2 min and reached a maximum within 5 min; RL remained above baseline at 20 min. Pretreatment with the bradykinin B2 receptor antagonist, Hoe 140, decreased the bronchoconstrictor effect of ovalbumin markedly at 10 to 20 min. In the presence of phosphoramidon (4.5 mumol kg-1, i.v.) the inhibition induced by Hoe 140 was apparent earlier and remained over the 20 min period of study. 4. Pretreatment with a combination of NK1 (CP-96,345) and NK2 (SR 48968) tachykinin receptor antagonists also markedly inhibited ovalbumin-induced bronchoconstriction; addition of the bradykinin B2 receptor antagonist to the NK1 and NK2 tachykinin receptor antagonists had no additional inhibitory effect on antigen-induced bronchoconstriction.5. These findings confirm that activation of sensory nerves to release tachykinins in guinea-pig airways contribute to antigen-induced bronchoconstriction, and provide evidence that tachykinin release is due to kinins generated during the allergic response.

The role of tachykinins released from sensory nerves in bronchoconstriction induced by antigen was studied in sensitized guinea pigs anesthetized with pentobarbital sodium and pretreated with atropine. The combination of NK2 (SR-48968) and NK1 (CP-96,345) tachykinin-receptor antagonists abolished the increase in total pulmonary resistance (RL) evoked by intravenous capsaicin but did not affect the response evoked by intravenous histamine. A small dose of aerosolized ovalbumin (OVA, 0.1%) produced a small increase in RL that was further increased and markedly prolonged by the neutral endopeptidase (NEP) inhibitor phosphoramidon; this bronchoconstrictor effect of OVA was markedly reduced by the NK2-receptor antagonist and was abolished by the combination of the NK1 and NK2-receptor antagonists together. When a larger dose of OVA (0.5%) was used, a maximal bronchoconstrictor response was obtained. Phosphoramidon did not potentiate this response significantly. The combination of NK1- and NK2-receptor antagonists blunted the response at 5 min only slightly but markedly attenuated the later (10-20 min) response. These results show that tachykinins released from sensory nerves play a significant role in antigen-induced bronchoconstriction in guinea pigs. This effect is exaggerated when the normal modulation of neuropeptides by NEP is inhibited and is mediated predominantly by NK2-receptor activation, with a smaller contribution by NK1 receptors.

The role of NK1 tachykinin receptors in the increase in total pulmonary resistance (RL) produced by release of endogenous tachykinins was investigated in anesthetized guinea pigs pretreated intravenously with atropine (1 mumol/kg) by using the novel nonpeptide antagonists of NK1 (CP-96,345) and NK2 (SR 48968) tachykinin receptors. SR 48968 (0.3 mumol/kg) and CP-96,345 (2 mumol/kg) given intravenously completely blocked the response to the selective NK2 receptor agonist [beta Ala8]neurokinin A(4-10) and NK1 receptor agonist [Sar9,Met(O2)11]substance P, respectively. The response to neurokinin A was reduced dose-dependently, but not abolished, by SR 48968, and it was completely prevented by a combination of SR 48968 and CP-96,345. The response to capsaicin was reduced, but not blocked, by SR 48968, and it was completely abolished by the combination of the two antagonists. The combination of SR 48968 and CP-96,345 did not affect the increase in RL evoked by histamine. Thus, NK1, as well as NK2, receptor activation contributes to the noncholinergic increase in RL evoked by capsaicin and ascribed to release of endogenous tachykinins from sensory nerves.

The neutrophil enzyme elastase is a potent secretagogue of airway secretory cells, and elastase is present in high concentrations in sputum of patients with hypersecretion (e.g., cystic fibrosis, bronchiectasis). Interleukin-8 (IL-8), a recently discovered cytokine with potent neutrophil chemotactic properties in vitro, is also found in the sputum of these patients. We used an isolated tracheal segment in dogs in vivo to study the effect of IL-8 in causing neutrophil accumulation, elastase release, and secretion (by measuring lysozyme concentrations) in the luminal superfusate. IL-8 caused a potent time-dependent neutrophil accumulation at between 3 and 6 h. The effect was significant at 10(-9) and maximum at 10(-8) M. No increase in free elastase, cathepsin G, or lysozyme was detected in the superfusate. Thus, in contrast to previous studies showing that ragweed antigen causes the accumulation of neutrophil elastase which in turn causes lysozyme secretion, IL-8 causes neutrophil accumulation without granule secretion (or subsequent secretagogue activity). The findings were confirmed with dog and human neutrophils in vitro.

We have investigated the ability of ruthenium red, an inorganic dye with Ca2+ entry-blocking properties and a selective antagonist of capsaicin, and of indomethacin, a cyclooxygenase inhibitor, to inhibit bronchial smooth muscle responses evoked by toluene diisocyanate in guinea pigs. Previous exposure of isolated guinea pig bronchi to ruthenium red significantly decreased the response produced by toluene diisocyanate. Further, the response to toluene diisocyanate was significantly decreased by pretreatment with indomethacin. These findings provide evidence that toluene diisocyanate-induced contractions of guinea pig bronchi are produced indirectly by generation of a prostanoid that activates capsaicin-sensitive afferents via a ruthenium red-sensitive mechanism.

This study was designed to evaluate the mechanism of action of toluene diisocyanate (TDI) and the role of endogenous neutral endopeptidase in modulating in vitro contractile responses to TDI in guinea pigs. TDI (0.01-1 mM) produced a concentration-dependent contraction of the guinea pig main bronchi. Sensory nerve desensitization with capsaicin greatly reduced and in some cases almost abolished TDI-induced contractions. The neutral endopeptidase inhibitor phosphoramidon significantly increased the contractile response to TDI. Pretreatment with the substance P antagonist (D-Arg1,D-Pro2,D-Trp7,9,Leu11)-substance P greatly reduced TDI-induced contractions. These results suggest that TDI activates the "efferent" function of capsaicin-sensitive sensory nerves and that neutral endopeptidase may play a role in modulating the response in guinea pigs.

Skin mast cells release the neutral protease chymase along with histamine during degranulation. To test the hypothesis that chymase modulates histamine-induced plasma extravasation, we measured wheal formation following intradermal injection of purified mast cell chymase and histamine into the skin of ragweed-allergic dogs. We found that chymase greatly augments histamine-induced wheal formation. The magnitude of the potentiating effect increases with increasing doses of chymase and becomes maximal approximately 30 min after administration. Injection of chymase without histamine does not evoke wheal formation. The chymase potentiation of histamine-induced skin responses is prevented completely by pretreatment with the H1-receptor antagonist pyrilamine, and is prevented by inactivation of chymase with soybean trypsin inhibitor, suggesting that both histamine and preserved catalytic activity are required for the effects of chymase. To examine the effects of histamine and chymase released in situ in further experiments, we measured wheal size following local degranulation of mast cells by intradermal injection of ragweed antigen or compound 48/80. We found that pretreatment with either soybean trypsin inhibitor or pyrilamine markedly reduces ragweed antigen- or 48/80-induced wheal formation, supporting the results obtained by injection of exogenous chymase and histamine. These findings suggest a novel and important proinflammatory role for chymase in modulating the effects of histamine on vascular permeability during mast cell activation.

Airway reflexes are difficult to study in conscious animals because associated changes in ventilation alter intrathoracic airway dimensions. By studying an isolated segment of extrathoracic trachea, we have overcome this problem. In each of 2 dogs, we created surgically an isolated tracheal segment just below the larynx, sealed at one end and tapered at the other to a 3-mm opening via a skin fistula. A chronic tracheostomy was also created near the thoracic outlet. We monitored intraluminal pressure (Pseg) of the isolated segment to reflect changes in smooth muscle tone. During anesthesia, with pentobarbital, gentle mechanical stimulation of the carina, deflation of the lungs, and asphyxia for one min increased Pseg (+9 to +/- 16 cm H2O). Lung inflation and alveolar hyperventilation decreased Pseg (-9 to -16 cm H2O). Five breaths of 2 per cent histamine aerosol increased Pseg (+5 cm H2O) when resting tone was normal. We also coated lumen of the isolated segment with tantalum powder and documented roentgenologically changes in the size of the segment that reflected changes in smooth muscle tone; constriction and dilation in response to asphyxia and lung inflation, respectively, were demonstrated directly by this technique. In conscious dogs, lung inflation decreased Pseg, and carinal stimulation increased Pseg. Instillation of lidocaine hydrochloride (Xylocaine) into the isolated tracheal segment blocked cough caused by mechanical stimulation of the segment, but carinal stimulation still caused constriction of the segment under these conditions which indicated that afferent, but not effrent parasympathetic innervation of the segment had been blocked selectively. Conversely, instillation of atropine sulfate into the isolated tracheal segment blocked constriction of the segment caused by carinal stimulation, but mechanical stimulation of the segment still caused cough under these conditions, which indicated that efferent, but not afferent parasympathetic innervation of the segment had been blocked selectively. We conclude that an innervated extrathoracic tracheal segment constricts and dilates via cholinergic pathways and is suitable for the study of airway reflexes in conscious dogs.

We anesthetized, paralyzed, and ventilated 32 dogs. In 16 dogs we measured total pulmonary resistance (RL) during inhalation of acetylcholine (ACh), serotonin (5-HT), and histamine (Hist) aerosols. Cooling both cervical vagi reduced the bronchoconstriction caused by 5-HT 64% (P = 0.001), reduced Hist-induced bronchoconstriction 17% (P = 0.003), and did not significantly reduce bronchoconstriction due to ACh. In seven dogs, we ventilated both lungs separately through a double-lumen catheter. Application of 5-HT to one lung increased the transpulmonary pressure amplitude in the homolateral but not in the contralateral lung. Cooling the homolateral vagus reduced this response 32% (P = 0.02). In nine dogs, we stimulated the peripheral ends of both cut cervical vagi before and during aerosol application of ACh, 5-HT, and Hist. ACh and Hist increased baseline RL 97 and 134%, respectively, without increasing the effect of vagal stimulation. 5-HT increased baseline RL only 27% but greatly augmented the effect of vagal stimulation (mean increase, 271%, P = 0.001). We conclude that 5-HT acts to potentiate vagal effects on airway smooth muscle via the efferent vagal pathway.

Recent evidence suggests that nonadrenergic airway relaxation may be controlled by vasoactive intestinal peptide (VIP). The magnitude and duration of smooth muscle relaxation in response to VIP may be influenced by rates of peptide degradation after release from efferent peptidergic neurons. To explore the potential role of mast cell mediators in modulating neural control of airway tone, we studied the effect of the mast cell proteases tryptase and chymase on airway smooth muscle relaxation induced by VIP in ferret airway. Tracheal rings precontracted by serotonin (10(-6) M) in a muscle bath were relaxed by VIP (10(-7) M). We found that protease-rich supernatant obtained by degranulation of dog mastocytoma cells reversed VIP-induced relaxation, as did highly purified tryptase and chymase incubated with the tracheal rings. Either enzyme completely reversed the effect of VIP, but tryptase was more potent than chymase, paralleling previous test tube observations on the relative rates of VIP cleavage by the two enzymes. Inhibitors of mast cell tryptase and chymase preincubated with the supernatant or with the purified proteases prevented reversal of VIP-induced relaxation. Mast cell proteases did not reverse the tracheal relaxation caused by the nonpeptide adrenergic agonist isoproterenol. These findings show that mast cell proteases tryptase and chymase counteract the smooth muscle relaxant effects of VIP in ferret trachea and suggest a potential role for the mast cell proteases in the modulation of nonadrenergic neural control of airway tone by VIP.

Critical narrowing of the dependent airways was examined in anaesthetized dogs with closed chests. Two techniques were used, (a) "closing" volume (CV), measured from the expired nitrogen plateau and (b) tantalum bronchography, to measure the calibre of airways of 3-8 mm diameter in the upper (UZ), middle (MZ) and lower (LZ) zones. The point of airway "closure" at the junction of phases III and IV of the alveolar plateau coincided with an inflection in the diameter-lung volume curve of the airways in the most basal zone of the lung. This was accompanied by a sudden large increase in the calculated resistance of the airways in LZ compared with UZ. Following stimulation of the vagi CV increased and there was increased narrowing of airways, particularly in LZ. The addition of 0.5% halothane to the anaesthetic abolished the effect of vagal stimulation on CV and on airway resistance.

"Closing volume" (CV) was measured by the single-breath oxygen (SBO2) test in six dogs (alloxan group) before and after alloxan 100-200 mg/kg iv) was injected. CV increased significantly (P less than 0.05) from 32 +/- 3.2% (base line) to 45 +/- 3.5 % in period 1 (0-30 min after alloxan), but vital capacity (VC), respiratory system pressure volume (PV) curves, and alveolar plateau slopes did not change. No radiologic evidence of pulmonary edema was demonstrated in two dogs studied in period 1. CV decreased to 20 +/- 3.9% during period 2 (30-80 min after alloxan) and was associated with tracheal frothing, decreased VC, changes in the PV curve, and alveolar plateau slope, as well as histologic evidence of severe pulmonary edema. CV was 29 +/- 3.0%, and there were no changes in VC, PV curves, or alveolar plateau slopes in 6 other dogs studied for 2 h (control group). CV increased during period 1 before pulmonary edema could be demonstrated by changes in VC, PV curves, or radiography, but in period 2 lung function was so altered that CV by the SBO2 technique gave no useful information.

To determine whether thromboxane A2 released from aggregating platelets increases the contractile response of airway smooth muscle to cholinergic nerve stimulation and, if so, what the mechanism of action is, we studied in vitro bronchial segments from dogs under isometric conditions. The contractile responses to electrical field stimulation at 30 s and 1 min after the addition of autologous platelets were increased by 11.1 +/- 3.2 (SD) and 20.7 +/- 5.4%, respectively, and were accompanied by the release of thromboxane A2. These effects were inhibited either by pretreatment of platelets with indomethacin or by addition of the thromboxane A2 receptor antagonist SQ 29548. Likewise, the thromboxane A2 mimetic U 46619, in subthreshold doses (i.e., insufficient to increase base-line tension), increased electrical field stimulation-induced contraction by 18.7 +/- 4.8%. The increase was greater in the presence of a concentration of physostigmine that did not cause spontaneous contraction and was blocked by SQ 29548 but not by hexamethonium or by phentolamine. Methacholine-induced contractions were unaffected by U 46619. These results indicate that aggregating platelets, by releasing thromboxane A2, increase the airway contractile response to neural stimulation probably by the accelerated release of acetylcholine.

In 18 open-chest dogs we obtained pressure-diameter (P-D) curves from tantalum bronchograms and pressure-volume (P-V) curves by plethysmography. After vagotomy most of the decrease in diameter with decreasing transpulmonary pressure (Ptp) occured below 10 cmH2O and there was no P-D hysteresis. Smaller airways narrowed more with decreasing Ptp than larger ones. Bronchodilators did not increase diameters after vagotomy (P less than 0.2). With vagi intact, diameters were smaller at all Ptp (P less than 0.01) and exhibited hysteresis, but the lung P-V curve was unchanged. Vagal stimulation narrowed airways further at all Ptp and hysteresis was marked. Smaller airways narrowed more with vagal stimulation than larger ones. Vagal stimulation did not change the deflation limb of the P-V curve but decreased inflation volumes slightly at all Ptp (P less than 0.01). We conclude that in vivo tone is vagal and that it affects the physical properties of airways, but not of lungs, making the airways remarkably independent from lung parenchyma.

We describe a new in vivo chemotaxis assay in the dog trachea using a double-balloon endotracheal catheter. When inflated, the two balloons isolate a segment of trachea, which is perfused through Silastic tubes using a peristaltic pump. After instilling a chemotactic agent, the perfusate is sampled periodically to permit characterization of the chemotactic response. We anesthetized four mongrel dogs and ventilated them mechanically through the double-balloon catheter. Two mediators, leukotriene B4 (LTB4) and 8S,15S-dihydroxyeicosatetraenoic acid (8,15-diHETE) were tested in each dog by perfusing the trachea with each mediator in Hanks' balanced salt solution (HBSS) containing ethanol and antibiotics. Aliquots were removed for differential cell counts at fixed time intervals over a 4-h period. Control experiments performed in each dog with the identical concentrations of ethanol and antibiotics in HBSS showed no cellular response before 180 min. At 240 min, the cell counts were 86 +/- 28 (SE) granulocytes/microliter (n = 4). In contrast, both LTB4 and 8,15-diHETE gave a significant cellular response at 120 min (309 +/- 125 and 141 +/- 41 granulocytes/microliter, respectively; P less than 0.05) but did not differ significantly from each other. These results suggest that both LTB4 and 8,15-diHETE can incite inflammatory responses in the dog trachea in vivo. Furthermore, the double-balloon catheter technique promises to be a useful in vivo chemotaxis assay.

To determine which part of the parasympathetic bronchoconstrictor pathway is most sensitive to depression by general anesthetics, we stimulated different parts of the pathway in dogs after initial anesthesia with chloralose and urethan and then after additional anesthetic drugs. We stimulated the entire reflex pathway by producing apnea or hypoventilation, the sensory pathway by electrically stimulating the proximal end of cut superior laryngeal nerves, and the motor pathway by stimulating the distal end of a cut cervical vagus nerve. Bronchoconstrictor responses to all stimuli were assessed with a bypassed tracheal segment. When no additional anesthetic was administered, responses to all stimuli increased with time. Small additional doses of anesthetics (thiopental, 1-5 mg/kg; pentobarbital, 1-2 mg/kg; amobarbital, 1-2 mg/kg; or chloralose, 10 mg/kg) decreased responses to reflex and sensory stimulation markedly and reversibly, but they did not affect responses to motor stimulation. Increased doses decreased responses to motor stimulation as well. Our previous study (Skoogh et al., Am. Rev. Respir. Dis. 123: 202, 1981) showed that barbiturates depress parasympathetic ganglionic synapses; the present study suggests that central nervous system synapses may be even more sensitive to depression by general anesthetics.

To determine whether gamma-aminobutyric acid (GABA) affects the contractile properties of airway smooth muscle and, if so, what the mechanism of action is, the authors studied guinea pig tracheal rings under isometric conditions in vitro. GABA and related substances, baclofen and muscimol, had no effect on the resting tension but reversibly depressed contractions induced by electrical field stimulation in a dose-dependent fashion, IC50 values (mean +/- S.E.) being 5.6 +/- 1.4 X 10(-6) M, 6.8 +/- 0.9 X 10(-6) M and 8.5 +/- 1.5 X 10(-5) M, respectively. In contrast, GABA did not alter the response to exogenous acetylcholine or the nonadrenergic noncholinergic inhibitory component. Pretreatment of tissues with bicuculline antagonized the inhibitory effect of GABA as well as that of baclofen. This inhibitory effect was not modified by propranolol, phentolamine, hemicholinium-3 or naloxone, but it was blocked by the Cl channel blocker furosemide and by the substitution of external Cl. These results suggest that GABA decreases the contractile response of airway smooth muscle to cholinergic nerve stimulation by inhibiting the evoked release of acetylcholine and that this effect is exerted by activating Cl-dependent, bicuculline-sensitive GABA receptors.

In seven studies on three dogs exercising on a treadmill (1.6 km/h), we studied the effect of ozone on ventilatory responses to hypercapnia and to hypoxia. After ozone exposure (0.67 +/- 0.02 ppm by vol; 2 h), the responses of minute volume of ventilation (VE) to progressive hypercapnia and hypoxia were not changed, but the breathing pattern in response to these stimuli changed. We analyzed the breathing pattern by plotting the relationship between VE and tidal volume (VT). During progressive hypercapnia, the slope of VE-VT relationship increased from a control value of 36.1 +/- 1.6 (mean +/- SE) to 93.5 +/- 8.9 min-1 after ozone (n = 7, P less than 0.005); during hypoxia, the slope increased from a control value of 46.1 +/- 8.6 to 142.7 +/- 18.3 min-1 after ozone (n = 6, P less than 0.005). The ozone-induced tachypneic responses to hypercapnia and hypoxia were not affected by inhalation of atropine sulfate or isoproterenol aerosols, but were completely abolished by bilateral vagal blockade. These findings indicate an effect of ozone on the vagal receptors located in the airways and lungs that causes reflex tachypnea during hypercapnia and hypoxia.

To determine the roles of endogenous enkephalinase (EC.3.4.24.11) in regulating tachykinin-induced contraction of airway smooth muscle, the authors studied the effects of the enkephalinase inhibitor leucine-thiorphan on the contractile responses to substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) in isolated ferret tracheal smooth muscle segments. Leucine-thiorphan shifted, in concentration-dependent fashions, the dose-response curves to all tachykinins to lower concentrations. Leucine-thiorphan changed the rank order of tachykinin potency from NKA greater than SP greater than NKB to NKA = NKB greater than SP. Removal of the epithelium slightly enhanced the contractile responses to SP and NKA but not to NKB. Atropine shifted the dose-response curves of all tachykinins to higher concentrations. Each tachykinin increased the contractile response to electrical field stimulation (5 Hz, 20 sec of duration, 20 V) in a dose-dependent fashion. This effect was not altered by hexamethonium, indomethacin, BW755C or naloxone but was potentiated by leucine-thiorphan and inhibited by the tachykinin receptor antagonist (D-Pro2, D-Trp7,9)-SP and by atropine. Because tachykinins did not affect contractile responses to acetylcholine significantly, their effects were probably on presynaptic postganglionic nerves. Captopril, bestatin and leupeptin did not alter contractile responses, suggesting that angiotensin converting enzyme, aminopeptidases and serine proteinases did not modulate tachykinin-induced effects. Enkephalinase immunofluorescence was found in the smooth muscle and epithelium and confirmed the authors' finding of enkephalinase-like activity in the muscle. The results suggest that tracheal enkephalinase is an important modulator of tachykinin-induced effects.

To study the roles of substance P and endogenous neutral endopeptidase in mediating cough, we measured cough responses in awake guinea pigs in response to exogenous substance P and capsaicin aerosols in the presence and absence of the neutral endopeptidase inhibitors leucine-thiorphan and phosphoramidon. Substance P stimulated cough in very low concentrations (10(-17)-10(-16) M). In a second study where the investigator did not know whether substance P or diluent alone was aerosolized, substance P (10(-16) M) caused cough. Leucine-thiorphan (10(-5) M) and phosphoramidon (10(-5) M) potentiated substance P-induced cough; NEP inhibitors also potentiated capsaicin-induced cough significantly. These findings suggest that substance P is a potent stimulator of cough responses, that capsaicin-induced cough is mediated by substance P or another similar neuropeptide, and that cough responses are modulated by endogenous neutral endopeptidase.

We studied whether the muscarinic antagonist, atropine, given intravenously or by inhalation, inhibits the bronchoconstrictor responses to inhaled acetylcholine and to acetylcholine released by electrical stimulation of the vagus nerves to the same degree. We assessed bronchoconstrictor responses in anesthetized dogs by determining the increase in total pulmonary resistance before and after increasing doses of atropine and then constructing inhibition dose-response curves. Before atropine the responses to the two stimuli were equal in magnitude. After intravenous atropine (initial dose 0.12 micrograms/kg, total dose 16 micrograms/kg) both responses were progressively inhibited to a similar degree. By contrast, after inhaled atropine (initial dose 0.02 micrograms/kg, total dose 2.4 micrograms/kg) the response to acetylcholine inhalation was inhibited to a much greater degree than the response to vagal stimulation. Thus, in studies designed to inhibit bronchoconstriction due to an inhaled muscarinic agonist to the same degree as bronchoconstriction due to a vagal reflex, atropine might better be given intravenously than by inhalation.

"Closing volume" (CV) was measured in 18 anaesthetized dogs before, during and after pulmonary vascular congestion produced by inflation of a balloon placed in the left atrium. In group A 10 dogs were anaesthetized with pentobarbitone. CV increased from 30.0 +/- 1.2 to 42 +/- 5.6% of vital capacity (P less than 0.05) when pulmonary wedge pressure was increased from 0.21 +/- 0.27 to 1.54 +/- 0.44 kPa (1.6 +/- 2.0 to 11.6 +/- 3.3 mm Hg) (P less than 0.05). In seven of these 10 dogs gas exchange was impaired after an increase of left atrial pressure. These changes in lung function during pulmonary vascular congestion were not associated with an increase in lung water. In group B, eight dogs were studied after anaesthesia with chloralose and urethane. Vagotomy or vagal cooling to 0 degrees C returned CV to baseline values in six dogs with pulmonary vascular congestion. In two dogs in which bilateral vagotomy had been performed before pulmonary vascular congestion there was no change in CV after balloon inflation. Isoprenaline abolished the effect of pulmonary vascular congestion on closing volume. This suggests that the vagus mediates the changes in lung mechanics associated with pulmonary vascular congestion.

In 4 unsedated, exercising dogs, we studied the effects of inhaled histamine aerosol on minute volume of ventilation, respiratory frequency, tidal volume, total pulmonary resistance, and dynamic pulmonary compliance. Inhalation (5 breaths) of 1 to 2 per cent histamine aerosols increased minute ventilation (mean, 50 per cent; p less than 0.001) by increasing respiratory frequency (mean, 166 percent; P less than 0.001), despite decreasing tidal volume (mean, 42 percent; P less than 0.0001). Total pulmonary resistance increased (mean, 200 per cent; P less than 0.001.) Breathing supplemental O2 did not affect the ventilatory response to histamine. Adding external resistive loads to a dog's airway did not simulate the pattern of rapid, shallow breathing produced by histamine. Inhalation of terbutaline prevented the changes in total pulmonary resistance and dynamic pulmonary compliance but did not alter the ventilatory response to histamine. When conduction in the cervical vagus nerves (which were implanted chronically in skin loops) was blocked by cooling, the ventilatory response to histamine was abolished. We concluded that histamine stimulates breathing by stimulation of receptors whose afferent pathways are in the vagus nerves; the effective stimulus is not bronchoconstriction but is presumably due to direct stimulation of airway receptors.

We studied the effect of vasoactive intestinal peptide (VIP) on the contractile responses to electrical field stimulation (EFS) in isolated ferret tracheal segments. VIP did not change resting tension up to 2 X 10(-7) M, but it showed a biphasic effect on the responses to EFS. In concentrations up to 10(-9) M, VIP potentiated the response; at higher concentrations VIP reduced responses. Thus, at a concentration of 10(-9) M, VIP decreased the mean (+/- SE) log EFS frequency, producing 50% of maximum contraction significantly from a control value of 0.476 +/- 0.062 to 0.214 +/- 0.057 Hz (P less than 0.01); at a concentration of 2 X 10(-7) M VIP increased the half-maximal frequency from a control value of 0.513 +/- 0.086 to 0.752 +/- 0.053 Hz (P less than 0.05). The potentiating effect of VIP (10(-9) M) was not inhibited by hexamethonium, indomethacin, pyrilamine, methysergide, or [D-Pro2,D-Trp7,9] substance P. The inhibitory effect of VIP (2 X 10(-7) M) was also not inhibited by hexamethonium, indomethacin, or naloxone. In contrast to EFS-induced contraction, contractions produced by acetylcholine (10(-9) to 10(-3) M) were not affected by VIP at concentrations of 10(-9) and 2 X 10(-7) M. These results suggest that VIP modulates contractions produced by EFS via presynaptic cholinergic mechanisms and probably through a specific VIP receptor.

Prostaglandin D2 (PGD2), the major product of arachidonic acid metabolism via the cyclooxygenase pathway in most mast cells, is present in the airways of atopic asthmatic patients after antigen challenge. Because eosinophilia is characteristic of asthma, we asked whether PGD2 causes eosinophils to accumulate in the airways in vivo. Using an endotracheal tube with two inflatable balloons we isolated a segment of trachea in four anesthetized mechanically ventilated dogs, and we superfused this segment with either a control solution (Hanks' balanced salt solution and antibiotics) or solution containing PGD2 (10(-6) M). Total and differential cell counts were determined at base line and every hour for 4 h during the study. PGD2 caused eosinophil accumulation in the trachea [7.0 +/- 3.4, 28.7 +/- 17.8, 33.7 +/- 13.6, and 35.4 +/- 10.7 (SD) cells/cm2 trachea after 1, 2, 3, and 4 h, respectively, P less than 0.05 vs. controls] but had no significant effect on neutrophil accumulation. The effect of PGD2 on eosinophil accumulation was significantly inhibited by the prostaglandin receptor antagonist SKF 88046 (5 mg/kg iv). We conclude that PGD2 is a selective stimulus that causes accumulation of eosinophils in the tracheal lumen of dogs in vivo.

During temporary left pulmonary artery occlusion (TLPAO) in dogs, blood in pulmonary capillaries downstream from the occlusion is static or flowing slowly. In such areas, the uptake of carbon monoxide (CO) and diffusing capacity (DLCO) should decrease with time as carboxyhemoglobin concentration increases. We measured DLCO during exhalation of five sequential breaths in anesthetized dogs using a modification of a technique recently described in our laboratory (J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 43: 617--625, 1977). During TLPAO, the decrease in DLCO was greatest at low lung volumes, suggesting that the occluded lung was emptying later in exhalation, a conclusion supported by measurements of DLCO during TLPAO with the right mainstem bronchus occluded. In addition. DLCO decreased with each breath as the backpressure to diffusion increased in static capillary blood. Inhalation of 4% CO accelerated the rate of decrease in DLCO. Measurement of DLCO during exhalation over multiple breaths may help detect pulmonary vascular obstruction.

The interaction between the effects of vagal stimulation and inhaled histamine on the bronchi was studied in anesthetized dogs. Reactivity was assessed by measuring changes in bronchial caliber visualized with tantalum bronchograms. In seven vagotomized dogs the bronchoconstrictor response to a combination of electrical stimulation of the vagus nerves and inhaled histamine solution produced a mean reduction in airway diameter (Daw) of 2.21 mm which was significantly greater than the additive results of the two stimuli applied separately (mean decrease in Daw 0.29 +/- 0.91 mm). In three dogs the effect of vagal stimulation was to produce a shift in the dose-response curve to inhaled histamine. These results indicate that the effect of the base-line bronchomotor tone must be considered in the evaluation of the effect of vagal blockade on airway reactivity. An increase in the resting degree of bronchomotor tone may contribute to the hyperreactivity observed in patients with asthma.

In five treadmill-exercising, unsedated dogs, we studied the effect of inhaled Ascaris suum antigen aerosols on minute volume of ventilation (VE), respiratory frequency (f), tidal volume (VT), total pulmonary resistance (RL), and dynamic pulmonary compliance (CLdyn), before and during cooling of the vagus nerves. With the vagi warm, inhaled antigen increased VE (mean + 62%; P less than 0.01)by increasing f (mean + 180%; P less than 0.01), despite a decrease in VT (mean - 42%; P less than 0.01). RL increased (mean + 170%; P less than 0.001) and CLdyn decreased (mean - 43%; P less than 0.005). With the vagi cool, inhaled antigen no longer affected VE, f, or VT (P greater than 0.5), although RL still increased and CLdyn still decreased. Inhalation of a bronchodilator, terbutaline, prevented the broncho-constriction induced by antigen but did not prevent the ventilatory response. We conclude that vagal afferent pathways mediate the ventilatory response to inhaled antigen and suggest that the primary stimulus for this response is not airway narrowing.

We studied the effect of a thromboxane A2-mimetic, U46619, on the contractile responses of canine bronchial smooth muscle to cholinergic stimulation in vitro. U46619 (3 X 10(-10) M), at a concentration that did not cause contraction, enhanced the effect of electrical field stimulation at all frequencies; histamine (10(-7) M) and prostaglandin F2 alpha (10(-7) M) did not. U46619 (3 X 10(-10) M) did not affect methacholine-induced contractions. U46619 may therefore increase the prejunctional release of acetylcholine.

Contractile responses of canine tracheal smooth muscle to electrical field stimulation diminished over a 2-h period of incubation. However, addition of indomethacin (10(-5) M) for a similar time not only prevented this inhibition of contractile response, but actually markedly increased the response to electrical field stimulation, suggesting that prostaglandins were responsible for the time-dependent inhibition. Measured prostaglandin E2 increased in the tissue bath over 2 h in control tissues. Addition of prostaglandin E2 to the tissue produced similar inhibition of contractile responses to electrical field stimulation in a concentration-dependent manner. In contrast, incubation alone, treatment with indomethacin, or addition of prostaglandin E2 had little, if any, effect on contractions induced by acetylcholine. We conclude that the release of prostaglandins from canine tracheal smooth muscle that occurs with time has a predominantly inhibitory effect on cholinergic neurotransmission at a prejunctional site.

To determine whether smooth muscle from airways made hyper-responsive by ozone exposure is hyper-responsive in vitro, tracheal smooth muscle strips taken from five dogs with airway hyper-responsiveness induced by ozone exposure were compared with strips from five control animals. On 1 day, airway responsiveness was assessed with dose-response curves of acetylcholine aerosol versus pulmonary resistance. On a second day, five dogs were exposed to ozone (3.0 p.p.m. for 2 h) and five were exposed to filtered air. Then airway responsiveness to acetylcholine was reassessed. All dogs were then killed, the trachea was rapidly removed and strips of smooth muscle were prepared from the central one-third of the trachea. The responsiveness of each strip to electrical field stimulation (contractions inhibitable by atropine and tetrodotoxin) and exogenous acetylcholine was assessed after 2 and 6 h of incubation and washing. Ozone caused a marked increase in responsiveness in vivo to acetylcholine with a fall in mean provocation concentration from 0.15 g % to 0.026 g % (P less than 0.001) while sham exposure had no effect. The responsiveness of muscle strips to electrical field stimulation in ozone-exposed dogs after 2 h of incubation and washing was increased when compared with 6 h of incubation and washing and with the control dogs (P less than 0.05 for EF50, the frequency of stimulation giving 50% maximum contraction). However, responsiveness to exogenous acetylcholine was similar in all strips from both ozone-exposed and control dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of lung macrophages stimulated with calcium ionophore on parasympathetic contractile response of canine bronchial rings was studied. Macrophages augmented the contraction induced by electrical field stimulation, an effect that was inhibited by indomethacin and by SQ29548, a thromboxane A2 receptor antagonist, but had no effect on the contractile response to exogenous acetylcholine. These results suggest that macrophage-derived thromboxane A2 facilitates cholinergic neurotransmission prejunctionally in airway smooth muscle.

We studied the effect of somatostatin on contractile responses to electrical field stimulation (EFS) in isolated ferret tracheal segments. Somatostatin (up to 10(-5) M) did not change resting tension, but it potentiated the contractile response to EFS dose dependently, with a maximum effect at 10(-6) M. Thus, at a concentration of 10(-6) M, somatostatin significantly decreased the mean log of EFS frequency producing 50% of maximum contraction from a control value of 0.52 +/- 0.07 to 0.24 +/- 0.06 (SE) Hz (P less than 0.01). The potentiating effect of somatostatin (10(-6) M) was not inhibited by hexamethonium, indomethacin, BW755C, pyrilamine, methysergide, or D,Pro2,D,Trp7,9-SP, but it was inhibited by atropine or by the somatostatin antagonist cyclo[7-aminoheptanoyl-Phe-D-Trp-Lys-Thr(Bzl)]. In contrast to EFS-induced contraction, contractions produced by acetylcholine (10(-9) to 10(-3) M) were not affected by somatostatin at a concentration of 10(-6) M. These results suggest that somatostatin potentiates contractions produced by EFS via presynaptic cholinergic mechanisms and probably through a specific somatostatin receptor.

To determine whether prostaglandin D2 (PGD2) modulates cholinergic neurotransmission in airway smooth muscle and, if so, what the mechanism of action is, we studied bronchial segments from dogs under isometric conditions in vitro. PGD2 (10(-8)-10(-5) M) elicited dose-dependent muscle contraction, which was reduced after blockade of muscarinic receptors, so that 50% effective dose (ED50) increased from 1.3 +/- 0.3 X 10(-6) to 3.9 +/- 1.0 X 10(-6) M by atropine (10(-6) M) (mean +/- SE, P less than 0.05). Physostigmine, at a concentration insufficient to alter base-line tension (10(-8) M), enhanced the PGD2-induced contraction and decreased ED50 to 6.4 +/- 0.5 X 10(-7) M (P less than 0.05). When added at the highest doses that did not cause spontaneous contraction (1.9 +/- 0.5 X 10(-7) M), PGD2 increased the contractile response to electrical field stimulation (1-50 Hz) by 21.9 +/- 6.6% (P less than 0.001). In contrast to this effect, the response to administered acetylcholine was not affected by PGD2. On the other hand, PGD2-induced augmentation of the response to electrical field stimulation (5 Hz) was further increased from 23.6 +/- 3.0 to 70.4 +/- 8.8% in the presence of physostigmine (10(-8) M) and was abolished by atropine but not affected by the alpha-adrenergic antagonist phentolamine or the histamine H1-blocker pyrilamine. These results suggest that the contraction of airway smooth muscle induced by PGD2 is in in part mediated by a cholinergic action and that PGD2 prejunctionally augments the parasympathetic contractile response, likely involving the accelerated release of acetylcholine at the neuromuscular junction.

To determine whether neutral endopeptidase (NEP), also called enkephalinase (EC 3.4.24.11), modulates the effects of exogenous and endogenous tachykinins in vivo, we studied the effects of aerosolized phosphoramidon, a specific NEP inhibitor, on the responses to aerosolized substance P (SP) and on the atropine-resistant response to vagus nerve stimulation (10 V, 5 ms for 20 s) in guinea pigs. SP alone (10(-7) to 10(-4) M; each concentration, 7 breaths) caused no change in total pulmonary resistance (RL, P greater than 0.5). Phosphoramidon (10(-4) M, 90 breaths) caused no change either in base-line RL (P greater than 0.5) or in the response to aerosolized acetylcholine (P greater than 0.5). However, in the presence of phosphoramidon, SP (7 breaths) produced a concentration-dependent increase in RL at concentrations greater than or equal to 10(-5) M (P less than 0.001). Phosphoramidon (10(-7) to 10(-4) M; each concentration, 90 breaths) induced a concentration-dependent potentiation of SP-induced bronchoconstriction (10(-4) M, 7 breaths; P less than 0.01). Vagus nerve stimulation (0.5-3 Hz), in the presence of atropine, induced a frequency-dependent increase in RL (P less than 0.001). Phosphoramidon potentiated the atropine-resistant responses to vagus nerve stimulation (P less than 0.001) at frequencies greater than 0.5 Hz. The tachykinin antagonist [D-Arg1,D-Pro2,D-Trp7,9,Leu11]-substance P abolished the effects of phosphoramidon on the atropine-resistant response to vagus nerve stimulation (2 Hz, P less than 0.005). NEP-like activity in tracheal homogenates of guinea pig was inhibited by phosphoramidon with a concentration producing 50% inhibition of 5.3 +/- 0.8 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutral endopeptidase (NEP) (enkephalinase, EC 3.4.24.11) and angiotensin converting enzyme (ACE) are two peptidases that can cleave the C-terminal dipeptide bradykinin(8-9) from bradykinin. To determine whether these peptidases play roles in modulating kinin-induced contractions in the airways, we studied the effects of captopril, an ACE inhibitor, and of leucine-thiorphan and phosphoramidon, two NEP inhibitors, on the contractile responses to bradykinin and lysyl-bradykinin in isolated segments of ferret trachea. Bradykinin and lysyl-bradykinin-induced contractions in a concentration-dependent fashion (P less than .001), with a threshold of 10(-7) M and 5 x 10(-7) M, respectively. In contrast, the bradykinin(8-9) and the N-terminal heptapeptide bradykinin(1-7), the major fragments of hydrolysis of bradykinin by NEP and ACE, had a very weak or no effect on tracheal contraction in concentrations as great as 10(-5) M. Captopril, leucine-thiorphan and phosphoramidon (each inhibitor at 10(-5) M, 15 min) shifted the concentration-response curves to lower concentrations by approximately 1 to 1.5 log U (P less than .05). Both NEP inhibitors and the ACE inhibitor potentiated the response to bradykinin in a concentration-dependent fashion (P = .0001), and the combination of phosphoramidon and captopril resulted in an additive potentiation of bradykinin-induced contraction (P less than .02). [D-Pro2-D-Trp7,9]-substance P, a substance P antagonist, did not modify the potentiation of bradykinin-induced contraction by NEP inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

To evaluate the role of airway neutral endopeptidase (NEP) in the regulation of contraction of airway smooth muscle in response to endogenous tachykinins, we studied the effects of the NEP inhibitor phosphoramidon on contractions of guinea pig bronchial smooth muscle strips induced by either electrical field stimulation (EFS) or by capsaicin. In the presence of atropine (10(-6) M), propranolol (10(-6) M), phentolamine (10(-5) M), indomethacin (10(-6) M) and pyrilamine (5 x 10(-6) M) EFS (biphasic; pulse width, 1.0 msec; frequency 0.5-5 Hz for 30 sec; intensity, 20 V) produced noncholinergic, nonadrenergic muscle contraction in a frequency-dependent fashion (P less than .001). Phosphoramidon potentiated the contractile responses to EFS (P less than .01). Leucine-thiorphan (10(-5) M), another NEP inhibitor, potentiated EFS-induced contraction in a similar fashion as phosphoramidon (186 and 182% of control, respectively; each comparison, P less than .025). Captopril, bestatin, leupeptin and physostigmine (each drug, 10(-5) M) were without effect (P greater than .5, N = 5). Capsaicin (1.5 x 10(-8) M) produced long-lasting atropine-resistant smooth muscle contraction, an effect potentiated by phosphoramidon (10(-5) M (P less than .001). Removal of the epithelium slightly but significantly (P less than .05) increased the contractile responses to capsaicin and to EFS at impulse frequencies of 2 and 5 Hz, and phosphoramidon substantially increased contractions in tissues without epithelium. The trachea, bronchi and lungs each contained significant NEP activity.(ABSTRACT TRUNCATED AT 250 WORDS)