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IODIZED oil (Ethiodol) infused directly into the lymphatic vessels permits radiographic visualization of remote lymph nodes in the pelvis and retroperitoneum and has proved to be valuable in the management of cancer involving the reticuloendothelial system.1 , 2 In a review of over 4000 patients examined by means of lymphangiography respiratory symptoms and clinical evidence of pulmonary vascular obstructions were rare.1 Although only 1 of our patients complained of "chest tightness" after the procedure the results of specialized pulmonary-function tests indicated transient obstruction of pulmonary capillaries in every case. Anatomic studies in dogs confirmed that oil microemboli lodged in pulmonary capillaries. Methods . . .

Exposure of humans to 4 to 6 ppM SO/sub 2/ decreased ratio of airway resistance to thoracic gas volume an average of 39% usually within 10 sec to 4 min. Atropine effectively blocked this change but did not decrease irritation or coughing during breathing. SO/sub 2/ delivered via tracheal cannula increased total pulmonary resistance in cats a mean of 246%. SO/sub 2/ delivered to upper airways only increased R/sub L/ a mean of 331%. Atropine or cooling of cervical vagosympathetic nerves negated this response. Rapidity of response and subsequent reversal suggests that changes in smooth muscle tone cause bronchoconstriction. Sensory receptors are in upper and lower airways, as lower airway constriction occurred with upper airway exposure. SO/sub 2/ acts like other stimuli producing bronchoconstriction.

Powdered metallic tantalum was used as a contrast agent for bronchography or tracheography in 15 studies on 13 patients (age, 13 days to 16 years). Tantalum proved to be an excellent medium, providing better mucosal detail and opacification than other contrast media, without obstructing airways. None of the patients had local or systemic reactions to tantalum. Because of its inertness and the small quantities required, tantalum may enable more extensive use of bronchography in the pediatric patient.

Eight patients were studied to determine the origin of their dyspnea. They had no clinical evidence of pulmonary hypertension and the pulmonary artery pressures were normal at rest. Special tests of pulmonary function showed that pulmonary vascular obstruction was present; the diagnosis was confirmed anatomically in five patients and by roentgenologic studies in two patients. In three patients pulmonary hypertension did not develop even during exercise; results of pulmonary angiograms and radioisotope scanning of the lungs in these patients were within normal limits. The discrepancy is explained by the presence of scattered lesions narrowing small pulmonary arteries and arterioles in these patients. The abnormal responses during pulmonary function studies included (1) increased V{inD}: V{InT}, which was present at rest in only two patients, but increased abnormally during exercise in all the patients in whom it was normal at rest; (2) decreased singlebreath diffusing capacity in every patient and decreased pulmonary capillary blood volume in every patient in whom the measurement was made; (3) partially compensated respiratory alkalosis at rest and during exercise in six of eight patients; and (4) significantly decreased arterial O 2 tensions during exercise in two patients (in one the hypoxemia was mainly due to right to left "shunting" of blood, and in one who exercised to high levels it was probably due to a diffusion defect). The abnormalities in pulmonary function not only allowed us to make the diagnosis but also were useful in following the course of the disease. Any patient with dyspnea during exertion who is suspected of having occlusive pulmonary vascular disease deserves detailed pulmonary function studies of the pulmonary circulation, including exercise tests, especially when pulmonary hypertension is absent and roentgenologic methods fail to demonstrate areas of large vessel obstruction.

Our aim was to determine whether flow from the submucosal glands of the trachea is reflexly regulated by sensory stimuli from the stomach in the cat, and, if such a gastropulmonary reflex exists, what sensory and motor pathways are important. We found that mechanical stimulation of the gastric mucosa increased submucosal gland secretions from 7.9 +/- 0.7 to 17.4 +/- 1.7 nl/min (mean +/- SE, P less than 0.001). This effect was prevented reversibly by cooling both abdominal vagus nerves to -3 degrees C before stimulation and was restored by rewarming the nerves. The effect was prevented irreversibly by cutting both abdominal vagus nerves and was then mimicked by electrically stimulating the central cut end of one of the nerves. This increase in secretions caused by electrical stimulation of the nerve was prevented by administration of atropine sulfate before stimulation. We conclude that stimuli from the stomach reflexly affect the rate of submucosal gland secretion. The sensory limb of this reflex lies in the abdominal vagus nerves, and the motor pathways are mediated by cholinergic muscarinic receptors.

We made concurrent measurements of the functional residual capacity (FRC) with the body plethysmograph (thoracic gas volume) and by 7-min and prolonged open-circuit nitrogen dilution methods (communicating gas volume). The mean difference between the 7-min communicating gas volume and the thoracic gas volume in 13 healthy subjects was only 0.13 liters. The thoracic gas volume averaged 0.99 liters larger than the communicating gas volume after 7 min of O2 breathing in 13 patients with emphysema. The communicating gas volume at 12–18 min was the same as the thoracic gas volume in 11 of 13 patients but was smaller in the other 2. When the thoracic gas volume was used to measure FRC, the total lung capacity averaged 142% of predicted normal in 13 patients with emphysema. Submitted on January 4, 1962

A procedure is described for producing a sulfur dioxide (SO2) contaminated atmosphere within a body plethysmograph, exposing man to this atmosphere while maintaining the SO2 concentration at a given level, and measuring the concentration with less than a oneminute lag time. A syringe is used to introduce incremental volumes of SO2 limiting the maximum SO2 concentration in the chamber and assuring safety of the subject. A Titrilog SO2 analyzer with its rapid response characteristics provides quick measurement of the SO2 concentration. The body plethysmograph used in this manner serves simultaneously as a pulmonary function measuring device and as an exposure chamber.

We measured airway resistance and thoracic gas volume by the body plethysmograph technique, and transpulmonary pressure in seven healthy, adult subjects, before and after induction of bronchoconstriction. A deep inspiration never altered airway resistance, measured at functional residual capacity in the control state, but always reduced it for 1—2 min when bronchoconstriction was present. Discrepancies in data published on airway resistance may be due to use of methods which require a deep inspiration, or to occurrence of a spontaneous deep inspiration shortly before the test. Submitted on January 13, 1961

Ion fluxes or fluid flow were measured across sheets of dog tracheal epithelium mounted in Ussing chambers or a special apparatus, respectively. Under short-circuit conditions, luminal amphotericin B (3 X 10(-5) M) caused an inhibition of net Cl secretion and an increase in net Na absorption across paired tissues. In paired tissues under resting open-circuit conditions, there was no significant net transepithelial flux of either Cl or Na. Amphotericin B induced significant net fluxes of both Cl and Na toward the serosal side. In separate tissues from the same animals, there was no significant transepithelial fluid movement under resting conditions. Amphotericin B caused a net absorption of fluid. The absorption of salt and fluid in amphotericin B-treated tissues was abolished by ouabain. We conclude that stimulation of active Na transport by amphotericin B leads to fluid absorption. In vivo, the movement of fluid across the dog tracheal epithelium may be dependent on a balance between active Cl secretion and active Na absorption.

To study the roles of substance P (SP) and endogenous peptidases in regulating mucus secretion from ferret trachea, we measured the SP-induced release of 35SO4-labeled macromolecules after incubating segments of trachea in Ussing chambers in the presence and absence of selected inhibitors of proteolytic enzymes. Our strategy was based on the idea that if endogenous peptidases degrade SP, then inhibitors of these enzymes should potentiate SP-induced secretion. We found that tracheas of ferrets contained SP-like immunoreactivity, and that SP stimulated the release of bound 35SO4 with rapid onset and offset. Eighty-five percent of the total macromolecular radioactivity released was contained in fractions of molecular weights greater than 10(6). The response to SP was concentration-dependent and reproducible. Thiorphan potentiated the secretory response to SP in a concentration-dependent fashion and phosphoramidon potentiated SP-induced secretion, whereas other inhibitors of proteinases and peptidases were without effects. These results suggest that substance P may regulate mucus secretion in ferrets, and that enkephalinase (dipeptidyl carboxypeptidase II, EC 3.4.24.11) in the airway degrades SP in a physiologically significant fashion, and thereby regulates peptide-induced secretion.

The production of airway secretions is important for protecting the airways. By active ion transport, airways can shift fluid across the epithelium. A discussion is presented of methods used to study the control of transepithelial fluid and electrolyte transport and how various agents modify this regulation.

We have investigated the activation of alpha-adrenergic contractile responses in dog tracheal smooth muscle. After cholinergic and beta-adrenergic blockade, neither electrical field stimulation nor alpha-adrenergic agonists caused contraction of trachealis strips in vitro, but after exposure to histamine or serotonin a striking contractile response was obtained. Similar activation of the contractile response to norepinephrine was seen in isolated tracheal segments in vivo after exposure to histamine and serotonin. This response was mediated predominantly by alpha 2-adrenoceptors, because the alpha 2-antagonist yohimbine was a potent inhibitor whereas the alpha 1-antagonist prazosin was a weak inhibitor of the response to both electrical stimulation and exogenous agonists. Using [3H]yohimbine to label alpha 2-receptors and [3H]prazosin to label alpha 1-receptors, we confirmed the preponderance of alpha 2-receptors in trachealis membranes but found no increase in either receptor number or affinity after incubating muscle strips with histamine. The magnitude of alpha-adrenergic contraction was significantly related to the magnitude of precontraction by histamine and serotonin both in vitro and in vivo but persisted after washout. Acetylcholine was much less potent in activating the alpha-adrenergic response. We conclude that activation of airway alpha-adrenergic responses involves a postreceptor mechanism not directly related to membrane depolarization, but involving some related process such as activation of calcium channels.

We isolated and characterized a chymotryptic serine proteinase from dog mastocytomas. Chymotryptic activity extracted at high ionic strength from mastocytomas propagated in nude mice was separated from tryptic activity by gel filtration and rapidly purified by sequential high-performance hydrophobic interaction and cation-exchange chromatography. The purified enzyme had an Mr of 27 000–30 000 by both analytical gel filtration and SDS-polyacrylamide gel electrophoresis, and a single amino-terminal sequence by automated Edman degradation. Like chymases from rat and human mast cells, the mastocytoma enzyme exhibited a high kcat/Km (1.1 · 105 M−1 · s−1) employing succinyl-l-Val-Pro-Phe-p-nitroanilide, the best of several p-nitroanilide substrates screened. It was inhibited by diisopropyl fluorophosphate and soybean trypsin inhibitor, but not by aprotinin, distinguishing it from the otherwise closely related neutrophil enzyme, cathepsin G. The amino-terminal 25 residues of mastocytoma chymase were found to be 72 and 68% identical to the corresponding sequences of chymases from rat peritoneal and mucosal mast cells, respectively; they were also closely related to human cathepsin G and to proteinase sequences from mouse cytotoxic T-lymphocytes. The mastocytoma chymotryptic enzyme contained an octapeptide sequence which from mouse is common to all chymotryptic leukocyte proteinases sequenced to date from four mammalian species; this feature distinguishes chymases and other chymotryptic leukocyte proteinases from serine proteinases of coagulation and digestion.

We determined the distribution of pulmonary alpha-adrenoceptors by autoradiographic localisation of [3H]prazosin binding to frozen sections of ferret lung. Specific binding of [3H]prazosin to lung sections was saturable and of high affinity (KD = 0.44 +/- 0.55 nM; mean +/- S.E., n = 5), with a specificity indicating binding to alpha 1-receptors. Autoradiographic showed that alpha 1-receptors were present in highest density in vascular smooth muscle (small vessels greater than large vessels), and were also present in airway submucosal glands and epithelium. There was also scanty labelling of alveolar walls which may be to contractile interstitial cells (Kapanci cells). Although smooth muscle of bronchi showed little labelling, surprisingly that of bronchioles was heavily labelled. The high density of alpha-receptors in small airways may be relevant to asthma in which alpha-adrenergic responses are activated. This method offers a means by which autonomic receptors of small airways may be investigated without the confusing contribution of other contractile elements and larger airways.

The site of greatest airway deformation in dog lungs was located during maximum expiratory flow by use of tantalum bronchography, fiberoptic bronchoscopy, and airway pressure measurements. A series of area vs. transmural pressure curves for each of these segments of the airway was produced after stepwise changes in transmural pressure. Measurements of area were made using cinephotography to elucidate the effect of time on airway compliance. The maximum flow rate was calculated using the t = 0.1 s compliance curve of the airway. An equation was derived so that maximum flow (V) could be calculated from the area (A) and transmural pressure (Ptm) of the flow-limiting segment. This equation, V = K-A square root of Ptm, implied that if V were constant then A must vary as Ptm-1/2. It was demonstrated that the area-transmural pressure curve of the flow-limiting segment showed this relationship between A and Ptm and that the flow calculated from this equation and the data from the A-Ptm curve gave flows identical to those measured during maximum expiration. The phenomena of effort-independent flow and negative effort dependence are also explained in terms of the area-transmural pressure curve of the flow-limiting segment.

A full-length cDNA encoding 15-lipoxygenase has been isolated from a human reticulocyte cDNA library. The predicted primary structure of the enzyme exhibits a sequence similarity of 61% and 45% with human 5-lipoxygenase and the soybean lipoxygenase isoenzyme I, respectively. When all three lipoxygneases are aligned, there are two distinct regions of significant sequence identity including a cluster of five histidine residues conserved in all three lipoxygenases. Because histidines can serve as ligands for the enzymatically active iron, this region may be critical to enzymatic function. These results provide a basis for exploring functional domains of lipoxygenases.

We anesthetized dogs, ventilated their lungs via the lower trachea, and exposed the epithelial surface of the upper trachea and coated it with powdered tantalum. Secretions from submucosal gland ducts formed elevations (hillocks) in the tantalum layer; we counted the number of hillocks that appeared in a 1.2-cm2 field. In 12 dogs, during normoxemia, 12 +/- 2 hillocks/cm2 formed in 90 s; during severe hypoxemia [fractional inspired O2 concentration (FIO2) = 0.05], 40 +/- 4 hillocks/cm2 formed in 90 s. Injections of sodium cyanide (25–75 micrograms) into the arterial supply to the carotid body also stimulated tracheal submucosal gland secretion. Secretory response to hypoxemia was suppressed by 1) section of both carotid sinus body nerves in six dogs and 2) section of both superior laryngeal nerves and vagus nerves in six other dogs. During mild hypoxemia (FIO2 = 0.10 or 0.15) tracheal submucosal gland secretion still increased. We conclude that hypoxemia increases secretion from submucosal glands in canine trachea by a carotid body chemoreflex.

Airway epithelium contains multiple mechanisms for affecting airway smooth muscle tone and responsiveness. Inflammatory diseases of the airways exhibit extensive changes in the epithelium (1, 2). Thus, alterations of airway epithelium may play important roles in the pathogenesis of bronchospasm in disease states.

An intravascular injection of substance P is known to increase vascular permeability in the rat trachea. Electrical stimulation of the cervical vagus nerve produces a similar response, presumably by releasing substance P or other tachykinins from sensory nerve endings. In the present study, we sought to determine whether the increase in vascular permeability induced by intravascular substance P or by vagal stimulation requires the presence of neutrophils or other components of circulating blood. To eliminate circulating blood, we perfused into the aorta of anesthetized rats an oxygenated Krebs-Henseleit solution containing albumin and monastral blue, a colloidal pigment that does not cross normal tracheal blood vessels. We then injected substance P intravascularly or electrically stimulated the right cervical vagus nerve. Increases in vascular permeability were quantified by using a microspectophotometer to measure the amount of extravasated monastral blue in tracheal whole-mounts. We found that the elimination of neutrophils and other components of circulating blood did not prevent the increase in tracheal vascular permeability induced by intravascular substance P or by vagal stimulation.

We measured the rate of fluid movement across the tracheal epithelium of the dog by an electrical method, modified from that originally described by Wiedner (Rev. Sci. Instrum. 47: 775-6, 1976). The base-line volume flow (Jv) from the submucosa to the mucosa was 44 +/- 23 nl.min-1.cm-2 (mean +/- SE; n = 6). In only three of the tissues was the resting Jv significantly greater than zero. Stimulation with 2 X 10(-3) M aminophylline (a drug that we have shown stimulates Cl secretion under short-circuit conditions) increased Jv in all tissues to a mean of 137 +/- 29 nl.min-1.cm-2 (P < 0.001). We also measured the unidirectional fluxes of Cl with the tissue at its spontaneous electrical potential. During the base-line period, the net flux of Cl was not significantly different from zero. During stimulation with aminophylline, Cl secretion increased to a mean of 1.44 +/- 0.50 microneq.cm-2.h-1. Comparison of the stimulated values of Jv and net Cl secretion suggests that fluid secretion by the trachea is isotonic during stimulation with aminophylline.

Lysozyme is a bacteriolytic enzyme found in respiratory tract fluid. In this study, immunocytochemistry was used to determine the cells of origin of tracheal lysozyme in the ferret. Lysozyme was found in secretory granules of serous but not mucous cells in the submucosal glands, and was absent from the surface epithelium, cartilage, and connective tissue. The exclusive presence of lysozyme in serous gland cells renders it useful as a biochemical marker of that cell type. Measurements of lysozyme assayed from the incubating medium indicated that bethanechol stimulated lysozyme release by 260 +/- 80.9% (mean +/- SE), phenylephrine by 80 +/- 16.4%, and terbutaline by 25 +/- 10.2%. Electron-microscopic and immunocytochemical analysis of incubated tissues revealed loss of serous granules and lysozyme immunoreactivity in response to the drugs. Atropine, propranolol, and phentolamine blocked the stimulatory effects of bethanechol, terbutaline, and phenylephrine, respectively. These findings establish the usefulness of lysozyme as a serous-cell marker and demonstrate that secretory responses of different magnitude are evoked by equimolar concentrations of alpha- and beta-adrenergic and cholinergic drugs.

The idea that the fluid component of cat tracheal submucosal gland secretions is produced by Na-linked secretion of Cl was tested. Gland secretion was stimulated with phenylephrine; gland fluid flow, net salt movement, and output of 35SO4-labeled macromolecules were measured. With CI, I, NO3, or Br as the major anion, phenylephrine caused equal increases in gland flow and output of 35S-labeled macromolecules while increasing net transepithelial 22Na movement from 0 to about 10 mu eq X cm-2 X h-1. With the impermeant gluconate as a major anion, phenylephrine caused the same increase in output of 35S label, a smaller increase in gland flow, and had no effect on net 22Na movement. Short circuiting in the presence of Cl, or high concentrations of the loop diuretics, furosemide or bumetanide, did not alter the actions of phenylephrine. Ouabain or replacement of Na by choline or Li abolished all secretory effects of phenylephrine. We conclude that active Cl secretion is not responsible for the transepithelial flows of salt and water induced by phenylephrine. Instead, these flows may be secondary to the release of osmotically active components of the secretory granules.

The mast cell proteases tryptase and chymase have long been known to constitute one-fifth of the total protein in mast cells. However, their biological functions have not been easy to study because of the difficulty in obtaining sufficient amounts of the enzymes to study their biological functions. Recently, we have been fortunate to have available a permanent line of dog mastocytoma cells to purify both enzymes to homogeneity, and we have used the purified enzymes in two ways. First, in a series of biological studies, we have discovered unique and potent actions of the enzymes that may provide important insights into the pathogenesis of diseases such as asthma and cystic fibrosis. Important biological activities are also likely to exist in other tissues. Because of their structures, mast cell proteases are likely to act in proximity to their sites of release. Thus, the presence and amounts of tryptase and chymase in specific loci may play important roles in tissue responses. In diseases such as asthma and cystic fibrosis, there is evidence that the expression of these mast cell enzymes changes, and these changes have important pathogenetic implications. Second, we have begun to perform structural studies of the enzymes. The recent cloning of tryptase by our group should assist in the better understanding of its functions. Crystallography of the pure proteins should provide further insights and could be the basis of rational development of potent and selective drugs that will inhibit their actions.

We studied the effect of a thromboxane A 2 -mimetic, U46619, on the contractile responses of canine bronchial smooth muscle to cholinergic stimulation in vitro. U46619 (3 × 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 F 2α (10 −7 M) did not. U46619 (3 × 10 −10 M) did not affect methacholine-induced contractions. U46619 may therefore increase the prejunctional release of acetylcholine.

Pulmonary epithelial cells may be primarily responsible for initiating or regulating inflammatory responses in the airways, in part by releasing chemical mediators. Among the most potent mediators of inflammation are the lipoxygenase metabolites of arachidonic acid, including the leukotrienes and the other mono- and dihydroxyeicosatetraenoic acids (HETEs). The human airway epithelium contains significant 15-lipoxygenase activity. Although some biologic functions of 15-lipoxygenase metabolites are known, further understanding of the role of this enzyme in the airway requires localization in tissue and studies of expression, regulation, and biologic activity. Towards these aims, we purified and characterized 15-lipoxygenase from eosinophil-enriched leukocytes. First, we studied cofactors that may be involved in regulating enzymatic activity. We discovered that calcium and phosphatidylcholine both enhanced, but ATP inhibited, the 15-lipoxygenase activity of highly enriched enzyme. Second, we isolated to homeogeneity, for the first time, human 15-lipoxygenase. This led to the determination of the N-terminal amino acid sequence and the discovery of homology among various mammalian lipoxygenases. Further research using purified lipoxygenase is expected to increase our understanding of the biologic roles and biochemical features of 15-lipoxygenation of arachidonic acid.

To determine the responsiveness of tracheal mucous cells to adrenergic and cholinergic stimulation, we analyzed changes in their structure induced by neurotransmitter-like agonists. Ferret tracheal rings were exposed for 30 min in vitro to one of the following: phenylephrine, isoproterenol, or bethanechol (all at 10(-5) M), in the presence of absence of appropriate antagonists. Electron microscopy and morphometric analysis revealed that the volume density of mucous cells (Vvmc, i.e. the space occupied by mucous cells in the submucosa) significantly decreased, and the surface density of mucous cell apical membrane (Svam) increased in response to isoproterenol and bethanechol but not to phenylephrine. In metabolic labeling experiments, the morphological changes were accompanied by secretagogue-evoked release of 35S-labeled macromolecules. Taken together, these data suggest that tracheal mucous cells secrete 35S-labeled macromolecules in response to beta-adrenergic and muscarinic agonists by an exocytotic process that involves a reduction in cell size.

To determine whether treatment with atropine causes dose-dependent inhibition of histamine-induced bronchoconstriction, we constructed dose-response curves to inhaled histamine after inhalation of placebo and 0.26 and 2.08 mg of atropine in eight subjects with mild asthma. Both doses of atropine significantly inhibited histamine-induced bronchoconstriction, and 2.08 mg caused significantly greater inhibition than 0.26 mg. Baseline specific airway resistance was significantly reduced by both doses of atropine but was no different after 2.08 mg than after 0.26 mg. There were considerable differences in the efficacy of atropine among individuals. We conclude that atropine causes dose-dependent inhibition of histamine-induced bronchoconstriction and that this effect is not merely a function of the atropine-induced in baseline airway caliber. The large magnitude of the atropine effect in some subjects and the small magnitude of the effect in others suggest that there is variability in the degree of involvement of muscarinic mechanisms in the exaggerated bronchomotor response to histamine in asthmatic subjects.

We sought to determine whether 0.25 ppm sulfur dioxide in filtered air causes bronchoconstriction when inhaled by freely breathing, heavily exercising, asthmatic subjects. Nineteen asthmatic volunteers exercised at 750 kilogram meters/min for 5 min in an exposure chamber that contained filtered air at ambient temperature and humidity or, on another day, filtered air plus 0.25 ppm sulfur dioxide. The order of exposure to sulfur dioxide and to filtered air alone was randomized, and the experiments were double-blinded. Specific airway resistance, measured by constant-volume, whole-body plethysmography, increased from 6.38 +/- 2.07 cm H2O X s (mean +/- SD) before exercise to 11.32 +/- 8.97 after exercise on days when subjects breathed filtered air alone and from 5.70 +/- 1.93 to 13.33 +/- 7.54 on days when subjects breathed 0.25 ppm sulfur dioxide in filtered air. The increase in specific airway resistance on days when subjects breathed 0.25 ppm sulfur dioxide was only slightly greater than on days when they breathed filtered air, but the difference was significant. To determine whether 0.25 ppm sulfur dioxide causes greater bronchoconstriction in asthmatic subjects exercising more vigorously, 9 subjects then repeated the experiment exercising at 1,000 instead of 750 kilogram meters/min. Specific airway resistance increased from 6.71 +/- 2.25 to 13.59 +/- 7.57 on days when subjects breathed filtered air alone and from 5.23 +/- 1.23 to 12.54 +/- 6.17 on days they breathed 0.25 ppm sulfur dioxide in filtered air. The increase in specific airway resistance on the 2 days was not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)

Our aim was to determine whether adrenergic as well as cholinergic nerves mediate secretion of fluids from tracheal submucosal glands and, if so, via which receptors. To do this, we studied the secretory responses of tracheal segments to electrical and pharmacologic stimulation in vitro in the presence and absence of a specific nerve blocker and autonomic antagonists. Stimulation caused small elevations, or "hillocks," the size of which we estimated by measuring their diameters. We found that electrical stimulation, acetylcholine, and phenylephrine each caused secretion but that terbutaline did not. Tetrodotoxin prevented the secretory response to electrical stimulation but did not prevent the responses to acetylcholine or phenylephrine. Neither atropine nor phentolamine alone prevented the response to electrical stimulation, but both drugs together did, and propranolol did not inhibit the adrenergic component of the response to electrical stimulation. Atropine blocked the response to acetylcholine, and phentolamine blocked the response to phenylephrine. We conclude that adrenergic and cholinergic nerves mediate secretion from the tracheal glands of ferrets via alpha-adrenergic and muscarinic receptors, respectively.

To follow up our previous observation that airway hyperresponsiveness induced by ozone is linked to airway inflammation, we investigated the effect of BW755C, an anti-inflammatory drug, on ozone-induced hyperresponsiveness in dogs. Airway responsiveness was assessed with dose-response curves of acetylcholine aerosol versus pulmonary resistance in two sets of experiments. In one set (placebo treatment), five dogs were given only saline solution treatment and were studied before treatment or ozone exposure and then after treatment both before and after ozone (3.0 ppm, 2 hours); in another set (BW755C treatment), the same dogs were studied before BW755C treatment or ozone and then after treatment (10 mg/kg intravenously) both before and after ozone. When the dogs were given no BW755C treatment, ozone induced a marked increase in airway responsiveness to acetylcholine. When the dogs were given BW755C, responsiveness was no different during treatment than before treatment but, more importantly, responsiveness did not increase significantly after ozone. We conclude that BW755C markedly inhibits ozone-induced airway hyperresponsiveness in dogs, probably by inhibiting the formation of oxygenation products of arachidonic acid.

Because leukotrienes have been implicated as putative mediators in upper airways disease, we studied whether leukotriene C4 (LTC4) might also have a mucus enhancing effect on submucosal glands. We anesthetized mongrel dogs with chloralose (100 mg/kg) and urethane (500 mg/kg) and ventilated them on a pump. To help visualize the secretions from submucosal glands, we exposed the mucosa of the upper trachea and coated its surface with powdered tantalum. Secretions from the glands (hillocks) were measured with time: The number of hillocks was measured at four time points on 19 dogs after each treatment in the sequence: no LTC4, LTC4, no LTC4 and LTC4 + blocker. The potential blockers were nerve cutting, atropine, FPL-55,712, and hexamethonium. Each potential blocker was used on 3-5 dogs. LTC4 was injected into the cranial thyroid artery. In 19 dogs with 27 responses, LTC4(8.6-11.0 micrograms) gave a positive response that was significantly different from control (P less than 0.01) at 1-4 min. These effects were not abolished in 5 dogs by cutting the superior laryngeal (SLN) and the vagus nerves (P less than 0.01). Pretreatment of the dogs (n = 5) with atropine, hexamethonium and the specific SRS-A (LTC4) antagonist FPL 55,712 (n = 3) gave a statistically significant (P less than 0.01-0.05) reduction in mucus secretion at all times for atropine, hexamethonium, and at all times except 4 min for (FPL 55,712). These results indicate that leukotriene C4 induces mucus secretion in dogs. This secretion does not depend on an intact reflex pathway but is altered at the individual gland by agents which block ganglionic motor pathways.

Extreme sensitivity of airways to multiple stimuli characterizes asthma. Airway hyperresponsiveness can be produced experimentally in otherwise healthy subjects or animals by inflammatory damage (e.g., induced by respiratory viruses or by inhaled oxidants). Evidence is presented that cell-to-cell interactions play an important role in experimental hyperreactivity and that similar inflammatory cascades may play a similar role in clinical asthma. Although the importance of epithelial cells and neutrophils has been identified in the present studies, other inflammatory mechanisms (e.g., sensory nerve release of substance P, epithelial mast cells, eosinophils) may also play key roles. In exercise-induced bronchospasm, the stimulus (e.g., cooling or drying) must affect a cell (e.g., one near the epithelial surface) by decreasing temperature or by increasing osmolality. This signal may cause mediator release and a subsequent cascade, leading to contraction of smooth muscle. Environmental irritants (e.g., ozone) inhaled during exercise may potentiate these effects by producing further inflammation.

Asthma is a disease of the airways characterized by reversible airway narrowing. Airway smooth muscle hyperresponsiveness to a variety of stimuli plays an important role in asthma. The clinical response in asthma depends on both the degree of the stimulus and the degree of responsiveness of the smooth muscle. Thus, stimuli that normally produce no clinical response can produce symptoms in the presence of smooth muscle hyperresponsiveness.

It has been well-known that exercise increases ventilatory variables in humans and in animals1,2 This “exercise-induced hyperpnea” are characterized as 1) quick response of ventilatory parameters, 2) isocapnic or hypocapnic hyperpnea and 3) disproportinately augmented ventilation compared to exercise-induced metabolism.

We studied the effect of prostaglandin F2 alpha (PGF2 alpha) on the responsiveness of pulmonary airways in dogs. Airway responsiveness was assessed by determining the bronchoconstrictor response to increasing concentrations of acetylcholine aerosol delivered to the airways. In each of five dogs, we determined responsiveness during treatment with physiologic saline, histamine, or PGF2 alpha aerosols. The doses of histamine and PGF2 alpha were determined by establishing the largest dose of each which could be given to the dog without causing bronchoconstriction (subthreshold doses). We found that airway responsiveness was not significantly different during histamine treatment than after saline, however, responsiveness increased during treatment with PGF2 alpha. In addition, the hyperresponsiveness induced by PGF2 alpha was prevented by pretreatment with the ganglion blocking drug hexamethonium (5 mg/kg given intravenously). The results show that PGF2 alpha specifically increases the responsiveness of pulmonary airways in doses that do not cause bronchoconstriction, and suggest that the hyperresponsiveness involves a neural mechanism such as increased responsiveness of airway sensory nerves.

β-Adrenergic agonists regulate a variety of lung functions including relaxation of airway smooth muscle, secretion of mucus1, release of surfactant2 and modulation of mediator release from mast cells3. Assays of the direct binding of radio-labelled β-adrenergic antagonists to homogenates of whole lung from several species have revealed a very high density of β-adrenoreceptors, but the cellular location of these receptors has not been determined4–7. Using a technique previously used to study distribution of receptors in the brain8,9 we report here for the first time the localization of β-receptors in the lung by light microscopic autoradiography. Initial biochemical studies showed that 3H-dihydroalprenolol (3H-DHA) bound to slide-mounted frozen sections of ferret lung with the characteristics expected of interaction with β-receptors. Autoradiograms prepared by apposition of dry emulsion-coated coverslips to these sections showed that the distribution of β-receptors in the lung was widespread. The highest concentration of β-receptors was found in smooth muscle of bronchioles with somewhat lower densities in smooth muscle of large airways. Labelling also occurred in airway epithelium, submucosal glands and vascular smooth muscle. Alveolar walls were heavily labelled and as type II pneumocytes, which are known to have β-receptors, comprise only a small portion of the surface area, β-receptors must also be present on type I cells and/or capillary endothelial cells, although their function is at present unknown.

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)

[3H]Quinuclidinyl benzilate binding to slide-mounted frozen sections of ferret lung was of high affinity ( K d 63 ± 14 pM , mean ± S.E. , n = 4 ), and characteristic of interaction with muscarinic cholinergic receptors. Light microscopic autoradiography showed muscarinic receptors to be localized predominantly to smooth muscle of trachea and intrapulmonary cartilaginous airways, and to submucosal glands. There was much less labelling of bronchiolar smooth muscle, airway epithelium and vascular smooth muscle and no labelling of alveoli. This distribution of receptors parallels that of cholinergic innervation.

To determine whether recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11) prevents cough induced by exogenously applied and endogenously released neuropeptides, we measured cough responses to aerosolized solutions of substance P or of capsaicin for 2 min in random-source guinea pigs before or after exposing them to aerosolized recombinant human enkephalinase. Substance P (10(-16) M) increased coughing compared with its vehicle. Enkephalinase (120 micrograms) inhibited cough induced by subsequent exposure to substance P compared with the response to substance P alone, but after further exposure to the enkephalinase inhibitor leucine-thiorphan (10(-5) M), substance P increased cough significantly. Similar results were obtained for capsaicin-induced cough. In pathogen-free guinea pigs, after they inhaled inactive recombinant enkephalinase (33 micrograms), capsaicin (10(-13) M) increased cough significantly. In contrast, after they inhaled active recombinant enkephalinase (33 micrograms), capsaicin increased cough only slightly. These results suggest that aerosolized enkephalinase reaches the sites of release or actions of endogenous neuropeptides and, by degrading them, prevents cough induced by their release. Furthermore, these studies suggest that recombinant enkephalinase might be useful in the treatment of cough and other symptoms of diseases involving peptides cleaved by this enzyme.