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To determine the tachykinin receptor subtype that mediates the increase in vascular permeability, we examined the rank order of potency of tachykinins for inducing plasma extravasation in guinea pig skin and the specificity of tachykinin-induced tachyphylaxis of the responses. Plasma extravasation of the skin induced by tachykinins was NK-1 (SP-P)-type response from the rank order of potency of mammalian and nonmammalian tachykinins. Tachyphylaxis of the vascular response was induced by intradermal preinjection of mammalian tachykinins and was tachykinin-specific. In substance P (SP) tachyphylaxis (where SP was preinjected), the response to SP, not to neurokinin A (NKA) or neurokinin B (NKB), was decreased. In NKA tachyphylaxis and NKB tachyphylaxis, the responce to NKA, not to SP or NKB, and the response to NKB, not to SP or NKA, were decreased, respectively. Thus, we conclude that the apparent NK-1-type response is mediated through three mammalian tachykinin receptors, NK-1, NK-2, and NK-3, which are specifically stimulated by their preferred agonist, SP, NKA, and NKB, respectively.

Bovine tracheal submucosal gland cells have been isolated by enzymatic digestion and serially propagated in tissue culture for more than 12 mo. (40 passages). The cells exhibit an epithelioid appearance at confluence and contain alcian blue (pH 2.5)/periodic acid-Schiff-positive material within cytoplasmic granules. By electron microscopy numerous osmiophilic secretory granules are seen. Maximal growth is observed when the cells are grown on human placental collagen-coated culture vessels in medium supplemented with 20% fetal bovine serum. Scintillation spectrometry revealed that radiolabeled precursor (35SO4) was incorporated into high molecular weight molecules and released from cells. Isoproterenol (10(-6) to 10(-3) M) stimulated the release of 35SO4. The maximal response to isoproterenol was completely inhibited by the beta-adrenergic antagonist propranolol. It is concluded that the cultured cells retain features of tracheal gland cells and may serve as a useful model of synthesis and secretion of macromolecules by tracheal gland cells.

The sections in this article are: 1 Airway Secretions 1.1 Surface Mucus 1.2 Submucosal Glands 1.3 Effects of Drugs and Mediators 1.4 Hypertrophy of Mucus-Secreting Cells 2 Ion Transport and Water Movement 2.1 Introduction 2.2 Mechanisms of Ion Transport 2.3 Regulation of Ion Transport 2.4 Relationship Between Ion Transport and Water Movement

An abnormally large number of mast cells in the airway lumen may be an important factor in the pathogenesis of bronchial hyperreactivity. However, it is unclear just how many mast cells are present in the lumen of normal or hyperreactive airways, in part because of differences in the histochemical techniques that have been used to identify mast cells and questions about the heterogeneity of mast cells. The present study was done (1) to compare the effectiveness of six techniques in the identification of mast cells obtained from dogs by bronchoalveolar lavage (BAL), (2) to compare the mast cells in the airways of normal mongrel dogs with those from a breed of dog (Basenji-Greyhound) known to have bronchial hyperreactivity, and (3) to determine whether the two-type histochemical classification used for rodent mast cells (formaldehyde-resistant or typical and formaldehyde-sensitive or atypical) applies meaningfully to the mast cells in BAL fluid from dogs. Cells obtained by BAL were fixed with Mota's basic lead acetate or formaldehyde. Mast cells were identified by metachromatic staining with toluidine blue or methylene blue, staining of highly sulfated proteoglycans with Alcian blue or berberine sulfate, and a histochemical reaction for chloroacetate esterase (mast cell chymase). After Mota's fixation, the methods were relatively similar in their effectiveness in determining the number of mast cells in lavage fluid from the mongrel dogs, in that all of the values fell within a narrow range: 0.53 to 0.96% of the total number of cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The sections in this article are: 1 Sympathetic Nervous System 1.1 Sympathetic Innervation 1.2 Circulating Catecholamines 1.3 β-Receptors 1.4 α-Receptors 2 Nonadrenergic Inhibitory Nervous System 3 Parasympathetic Nervous System 3.1 General Considerations 3.2 Altered Parasympathetic Activity 3.3 Studies of Asthmatic Patients 4 Conclusion

Airway epithelial cells produce mediators that play a role in regulating airway smooth muscle function. This study was designed to examine the effects of epithelial-derived products on contraction of airway smooth muscle. To avoid biochemical and physical changes that may be produced by stripping epithelium from tracheal smooth muscle, we examined the effect of products from pure cultured tracheal epithelial cells on intact dog tracheal smooth muscle. When bradykinin (10(-5) M) was added to dog epithelial cells in culture and the supernatant was added to strips of isolated tracheal smooth muscle, contractile responses to electrical field stimulation were significantly inhibited. Pretreatment of the epithelial cells with indomethacin (5.6 x 10(-6) M) inhibited this effect. Bradykinin placed directly on the canine smooth muscle had no effect on resting tension or on the response to electrical field stimulation. Contractions of the smooth muscle to exogenous acetylcholine were unaffected by supernatants from either indomethacin-treated or untreated cells stimulated with bradykinin (10(-5) M) compared to time controls. We conclude that bradykinin stimulates the release of a cyclooxygenase-dependent inhibitory factor from airway epithelial cells. This factor is likely to be prostaglandin E2, which is generated by the epithelial cells in response to bradykinin stimulation and inhibits smooth muscle contraction induced by electrical field stimulation. Although the mechanism of this inhibition is unknown, the normal response to exogenous acetylcholine is consistent with the hypothesis that prostaglandin E2 acts by inhibiting cholinergic neurotransmitter release at a prejunctional site.

We studied the effect of vasoactive intestinal peptide (VIP) on the output of 35S-labeled macromolecules from ferret tracheal explants either placed in beakers or suspended in modified Ussing chambers. In Ussing chamber experiments, the radiolabel precursor, sodium [35S]sulfate, and all drugs were placed on the submucosal side of the tissue. Washings were collected at 30-min intervals from the luminal side and were dialyzed to remove unbound 35S, leaving radiolabeled macromolecules. Vasoactive intestinal peptide at 3 × 10−7 M stimulated bound 35S output by a mean of +252.6% (n = 14). The VIP response was dose-dependent with a near maximal response and a half maximal response at approximately 10−6 M and 10−8, M, respectively. The VIP effect was not inhibited by a mixture of tetrodotoxin, atropine, l-propranolol, and phentolamine. Vasoactive intestinal peptide had no effect on the electrical properties of the tissues. We conclude that VIP stimulates output of sulfated-macromolecules from ferret tracheal sub...

We studied the effects of neurokinin A (NKA) and neurokinin B (NKB), the mammalian-derived tachykinins, on the electrical and ion transport properties of canine tracheal epithelium. Both tachykinins dose-dependently increased short-circuit current (Isc) when added to the mucosal (NKA: delta Isc(max) = 24.2 +/- 2.4 microA/cm2, KD = 9 nM; NKB: delta Isc(max) = 1.42 +/- 2.2 microA/cm2, KD = 32 nM) or submucosal (NKA: delta Isc(max) = 10.5 +/- 1.2 microA/cm2, KD = 45 nM; NKB: delta Isc(max) = 2.2 +/- 1.4 microA/cm2, KD = 80 nM) bath. Isc responses to mucosal addition of tachykinins consisted of transient and subsequent steady-state components, whereas submucosal addition elicited only steady-state responses. Inhibition of Cl transport with bumetanide or substitution of Cl reduced the maximal changes in Isc. In paired tissues, NKA increased net 36Cl flux toward the mucosa from 1.83 +/- 0.49 to 2.71 +/- 0.46 mu eq.cm-2.h-1 (p less than 0.05), without affecting net 22Na flux toward the submucosa. The increases in Isc induced by tachykinins were not modified by prior tissue incubation with phentolamine, propranolol, atropine, tetrodotoxin, or indomethacin, but were effectively inhibited by (D-Pro2, D-Trp7,9)substance P. The cyclic AMP (cAMP) levels in the surface epithelium were increased by the addition of NKA and NKB. These findings suggest that NKA and NKB selectively stimulate the secretion of Cl across canine tracheal epithelium, probably by acting directly on the tachykinin receptors, and that these effects are associated with the increased production of intracellular cAMP.

Summary Globule leukocytes in the epithelium of the rat trachea may be counterparts of mucosal mast cells that are located in the gastrointestinal tract. If they are indeed similar to mucosal mast cells, globule leukocytes would be expected to decrease in number in rats treated with dexamethasone but not in rats treated with compound 48/80, an agent which causes non-antigenic degranulation of connective tissue mast cells. In this study, we determined the number and compared the distribution of globule leukocytes and connective tissue mast cells in the tracheas of pathogen-free rats. We then determined whether the number of these two types of cells changes in rats treated for 5 days with compound 48/80, dexamethasone, a combination of compound 48/80 and dexamethasone, or saline. We identified globule leukocytes and mast cells in whole mounts and histological sections of rat tracheas by using a histochemical reaction that demonstrates the chymotrypsin-like protease (chloroacetate esterase) present in mast cell granules. Using this method, we found that aproximately 225000 globule leukocytes were present in the epithelium of the trachea. These cells were most abundant in the rostral trachea. Rats treated with dexamethasone had a 91% reduction in the number of globule leukocytes with protease-containing granules, but rats treated with compound 48/80 had a normal number of these cells. We found some 55000 connective tissue mast cells in the same tracheas. Mast cells were most abundant in the posterior membrane of the caudal trachea and in the lamina propria between cartilaginous rings. Rats treated with compound 48/80 had a 96% reduction in mast cells with protease-containing granules, but rats treated with dexamethasone had a normal complement of mast cells. We conclude that globule leukocytes are abundant in the tracheas of healthy rats, are similar in morphology and pharmacological responses to mucosal mast cells located in other organs of rats, and are more numerous than and have a different distribution than connective tissue mast cells. Globule leukocytes in the tracheal epithelium may have a role in respiratory defenses similar to that of mucosal mast cells in other organs.

To determine whether neutral endopeptidase regulates the binding of substance P to the receptors, and if so, what the mechanism is, we determined the effect of neutral endopeptidase inhibitors, thiorphan and phosphoramidon, on specific binding of 3H-substance P to homogenates of rat ileum. Specific binding was of high affinity and was saturable (dissociation constant, KD = 2.4 +/- 0.17 nM and number of maximal binding sites, Bmax = 101.1 +/- 5.5 fmol/mg protein), and the receptor subtype was substance P-P type. Neutral endopeptidase inhibitors increased the specific binding to up to 160% of control (P less than 0.005). Neutral endopeptidase inhibitors prevented the degradation of 3H-substance P during the binding assay and increased the amount of 3H-substance P remaining in the assay system to up to 4.5-fold of control (P less than 0.005), but did not significantly change the KD or Bmax values of specific binding. Protease inhibitors of kininase II, serine proteinases, or thiol proteinases did not significantly change either specific binding or the amount of 3H-substance P remaining in the assay system. We conclude that neutral endopeptidase regulates the binding of substance P to the receptors and that it does so by decreasing the amount of substance P available to the receptors, without significantly changing the affinity or the number of receptors.

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.

The incubation of suspensions of canine tracheal epithelial cells of greater than 95% purity with arachidonic acid (25–200 μg/ml) for 60–120 min resulted in the generation of a maximum of 36.2 ± 9.1 picomoles of leukotriene B 4 10 6 cells, less than 2.0 picomoles of leukotrienes C 4 , D 4 , and E 4 10 6 cells, and 1030 ± 463, 767 ± 500, and 324 ± 100 picomoles/10 6 cells of 15-, 12-, and 5-hydroxy-eicosatetraenoic acids, respectively (mean ± SEM, n = 8). The identity of leukotriene B 4 was established by chromatographic and spectral properties, by reactivity with mono-specific anti-plasma, and by the chemotactic activity for neutrophils. Thus, the epithelium may be an important source of mediators of inflammation and hypersensitivity of pulmonary airways.

Studies of the cellular effects of respiratory viruses have generally used cultures of non-airway (particularly renal) epithelial cells. This requires the assumption that, despite the marked differences between renal epithelium and airway epithelium, the virus-host cell interactions in cultures of renal epithelium will be relevant to those in airway epithelium. To study viral infection of airway epithelial cells, we removed the epithelial cells from ferret tracheas using 0.1% pronase solution, and plated them at a density of 5 X 10(5) cells/cm2 in collagen-coated plastic tissue culture wells. Cultures grew to confluence after 5-7 days. Viral inocula, consisting of supernatants from parainfluenza type 1-infected rhesus monkey kidney cell monolayers, were added to the culture medium in a concentration 10(3) times that sufficient to produce infection in 50% of rhesus monkey kidney monolayers (TCID50). Cytopathic changes, consisting of cellular elongation and detachment, became apparent after 3-6 days, at which time the medium contained 5 X 10(8) TCID50/ml. The monolayer appeared to be uniformly infected as revealed by adsorption of guinea pig erythrocytes. Specific immunofluorescence revealed uniformly positive staining for parainfluenza type 1 antigens. The ability to infect pure cultures of airway epithelial cells with viruses will allow us to examine the effects of these viruses on epithelial cell function, and to study virus-host cell interactions in cell cultures derived from the natural host cell.

We determined whether ozone-induced bronchial hyperresponsiveness in the rat is accompanied by neutrophil influx or increased vascular permeability in the trachea. Three groups of female Long-Evans rats were studied. One group was exposed to 4 ppm ozone for 2 h and studied immediately thereafter, another group was similarly exposed but was not studied until 24 h after the ozone exposure, and a third group consisted of control rats that breathed room air. Increases in total pulmonary resistance caused by acetylcholine aerosol were measured to assess bronchial responsiveness in these 3 groups. In parallel studies, neutrophil influx into the tracheal mucosa was quantified by counting cells within whole mounts of tracheas that were treated histochemically to stain the myeloperoxidase in neutrophils, and tracheal vascular permeability was quantified by measuring the amount of Evans blue dye extravasated into the trachea. In the rats studied immediately after the ozone exposure, the concentration of acetylcholine required to increase total pulmonary resistance to three-fold the baseline value was only 6% of that required in the controls. In the rats studied 24 h after the ozone exposure, this provocative acetylcholine concentration was not significantly different from that of the controls. Neither the number of neutrophils in the tracheal mucosa nor the amount of Evans blue dye extravasated into the trachea was significantly different from the corresponding control values at either time. We conclude that rats exposed to ozone develop bronchial hyperresponsiveness without detectable neutrophil influx or increased vascular permeability in the trachea.

To determine whether glycoconjugates can be released into airways by surface epithelial cells that do not contain secretory granules and, if so, whether extracellular proteinases can affect this release, we studied dog tracheal epithelial cells after 8-10 days in culture. Ultrastructurally, these cells showed an extensive cell surface coat and no secretory granules. Cells were pulse labeled with radioactive sulfate (Na2 35SO4, 50 microCi/ml/24 h) and washed free of the unbound label. Release of sulfated products was then measured at 20-min intervals under basal conditions and again after 20 min of incubation with various extracellular proteinase. We found that these cells synthesized sulfated products and released them spontaneously and continuously into the medium. In addition, trypsin, Pseudomonas aeruginosa elastase, thermolysin, Staphylococcus aureus proteinase, mast cell chymase, plasmin, and kallikrein (each at 10(-7) M except plasmin, at 5 X 10(-6) M) increased the release of sulfated products to 77-667% over baseline release (p less than 0.01, n = 5 dogs for each); preliminary results showed that human neutrophil elastase was also very potent. The sulfated products released by trypsin had an apparent molecular weight of greater than or equal to 10(6) da as determined by gel filtration on Sepharose Cl-4B. Over 50% of these 35S-labeled products were digested to low-molecular-weight products (500-2000 da) upon incubation with endo-beta-galactosidase or with keratanase, suggesting that they are glycoconjugates containing poly(N-acetyllactosamine)-type carbohydrate chains. Decrease in cell staining by lectins specific for poly(N-acetyllactosamine), which accompanied the release of glycoconjugates, indicates that these sulfated glycoconjugates were released by proteinases from the apical cell surface. We conclude that cultured tracheal epithelial cells synthesize and transport sulfated macromolecular glycoconjugates to apical cell surfaces. These glycoconjugates are released from cell surfaces when exposed to extracellular proteinases. We therefore suggest that macromolecular glycoconjugates in airway secretions can originate not only from secretory granules but also from epithelial cell surfaces during airway inflammation.

We studied the metabolism of sulfated cell-surface macromolecules in dog tracheal epithelial cells in primary culture. To examine the time-course and rate of appearance of sulfated macromolecules at the cell surface, the cells were pulsed with 35SO4 for short periods (5–15 min), and the incubation medium was sampled for spontaneously released macromolecules (basal secretions) and for release induced by trypsin (trypsin-accessible secretions). Trypsin-accessible 35S-labeled macromolecules appeared on the cell surface within 5–10 min, increased linearly, and plateaued by 40 min; the median transit time for 35S-labeled macromolecules to reach the cell surface was 21 min. 35S-labeled macromolecules in basal secretions increased with a similar time-course, reaching a plateau by 40 min. Incorporation of [3H]serine into the protein moiety of trypsin-accessible macromolecules occured more slowly; trypsin-accessible 3H-labelled macromolecules were barely detectable at 1 h and increased to a maximum after 2 h, suggesting the presence of a preformed pool of nonsulfated core protein. Pretreatment with cycloheximide, an inhibitor of protein synthesis, decreased trypsin-accessible 35S-labeled macromolecules log-linearly depending on the duration of pretreatment providing an estimate of the rate of depletion of the core protein pool ( t 1 2 = 32 min ). During continuous exposure to 35SO4, 35S-labeled macromolecules accumulated on the cell surface (trypsin-accessible compartment) for 16 h, at which point the cell-surface pool was saturated ( t 1 2 = 7.5 h ). After pulse-labeling the cells with 35SO4 for 15 min, the 35S-labeled macromolecules disappeared continuously from the cell surface ( t 1 2 = 4.6 h ), and 79% of the radioactivity was recovered in the medium as nondialyzable macromolecules. Release of the 35S-labeled macromolecules from the cell surface was abolished at 4°C, indicative of an energy-dependent process, but multiple proteinase inhibitors did not affect the release. We conclude that sulfate is metabolized rapidly into epithelial cell-surface macromolecules, which accumulate continuously into a relatively large cell-surface pool, before they are released by an undefined energy-dependent mechanism.

Histamine can be recovered from the blood of ragweed-sensitized dogs after aerosol antigen challenge, although its source is unknown. Neutrophils and eosinophils have been recovered from bronchoalveolar lavage fluid (BALF) obtained under identical conditions. We investigated the time course of changes in histamine levels in plasma and BALF taken from ragweed-sensitized dogs after aerosol challenge. Changes in the numbers of circulating neutrophils, eosinophils, lymphocytes, monocytes, and platelets were also studied. After 3 min, total pulmonary resistance (RL) was maximally increased and systolic blood pressure was maximally decreased. Histamine levels in plasma and BALF were increased and circulating eosinophils and neutrophils were decreased. After 15 min, platelet numbers were reduced. By 90 min, changes in RL, blood pressure, plasma and BALF histamine concentrations, and circulating neutrophils and eosinophils had returned to base-line values, but platelet numbers remained significantly decreased. Sham challenge caused no significant changes in any of these variables. Intravenous administration of histamine in doses large enough to attain plasma levels comparable with those achieved after aerosol antigen challenge resulted in no concomitant rise in BALF histamine levels. We conclude that antigen challenge in sensitized dogs causes increases in BALF and plasma histamine levels and is associated with a reduction in circulating neutrophils, eosinophils, and platelets. It is likely that antigen causes airway mast cells to release mediators that move down a concentration gradient from the airways to the pulmonary circulation.

A study to determine whether the bronchoconstriction induced by low concentration of sulfur dioxide in subjects with asthma decreases with repeated exposure was undertaken. Eight subjects with asthma performed 3 min of voluntary eucapnic hyperpnea with 0.5 ppm of SO2 in humidified filtered air three times at 30-min intervals and we measured specific airway resistance (SRaw) before and after each period of hyperpnea. Specific airway resistance increased significantly more after the first exposure to SO2 [(from 7.6 +/- 1.7 to 15.5 +/- 2.0 L x cm H2O/liter/sec (mean +/- SEM)] than after the second (from 8.1 +/- 1.3 to 10.8 +/- 1.6) or third (from 7.6 +/- 1.6 to 10.1 +/- 1.9) exposures (P less than 0.025). When seven subjects repeated hyperpnea with SO2 24 hr and 7 days later, SRaw increased as much as it had after the first exposure (from 8.2 +/- 2.5 to 15.5 +/- 4.5 at 24 hr and from 6.6 +/- 1.4 to 15.4 +/- 2.1 at 7 days). In four subjects repeated exposure to SO2 caused short-term inhibition of the bronchomotor response to SO2 but did not inhibit the bronchomotor response to histamine aerosol. It was concluded that repeated exposures to a low concentration of SO2 over a short period (on 1 day) can induce tolerance to the bronchomotor effects of SO2 in subjects with asthma. Tolerance to the bronchomotor effects of SO2 is not caused by decreased responsiveness of airway smooth muscle or a generalized decrease in the responsiveness of vagal reflex pathways since the bronchomotor response to histamine is preserved.

We exposed two awake dogs with a chronic tracheostomy and the cervical vagus nerves exteriorized in skin loops to 1.0 ppm of ozone (O3) for 2 h at intervals of 4 wk. We measured ventilatory variables before and after O3 exposure during rest and exercise before and after vagal block. We compared the effects of vagal blockade, exercise, and O3 on the primary determinants of breathing pattern (VT/TI, VT/TE, TI, and TE) in each of three conditions: base line (steady state), during hypercapnia, and after inhalation of 1% histamine. Under base-line conditions, O3 increased respiratory rate and decreased tidal volume (VT) by shortening time of expiration (TE) and time of inspiration (TI) without affecting VT/TI, an indicator of the neural drive to breathing. During progressive hypercapnia, O3 shortened TE and TI by effects both on tonic (nonvolume-related) and on phasic (volume-related) vagal inputs, and only the latter were prevented completely by cooling of the vagus nerves. Histamine-induced tachypnea was increased by O3 and was totally blocked by cooling the vagus nerves. We conclude that O3 shortens the timing of respiration without increasing ventilatory drive, shortens TI and TE through vagal and nonvagal pathways, increases tonic nonvagal and phasic vagal inputs, and stimulates more than one vagal fiber type.

To determine the effect of Neutral red (0.01%) on neural transmission through ganglia, we used an in vitro nerve-muscle preparation of ferret trachea. Before, during, and after incubating the trachea in Neutral red, we induced isometric muscle contractions first by activating preganglionic fibers with electrical stimulation of the vagus nerve, and then by activating postganglionic nerve fibers with electrical field stimulation. Incubation in Neutral red (0.01%) for 45 min at 38°C or 20°C reduced the responses to both pre- and postganglionic activation. When the control responses to pre- and postganglionic activation were matched, Neutral red depressed the 2 responses to the same degree, implying that the depression was confined to postganglionic structures. Washout of Neutral red from the medium restored the responses to both pre- and postganglionic activation. Histologic examination of all tissues proved that the ganglia were still stained after the washout procedure. We conclude that Neutral red (0.01%) depresses smooth muscle contractions evoked through neural pathways, and that this depression is reversible and confined to postganglionic structures, leaving ganglionic transmission intact.

A tryptic protease with the characteristics of a mast cell tryptase was purified from dog mastocytoma cells propagated in nude mice. Partial amino acid sequence of the mastocytoma tryptase revealed unexpected differences in comparison with other mast cell and leukocyte granule protease sequences. Extraction from mastocytoma homogenates at high ionic strength, followed by gel filtration and benzamidine affinity chromatography yielded a product with several closely spaced bands (Mr 30,000-32,000) on gel electrophoresis and a single N-terminal sequence. Nondenaturing analytical gel filtration revealed an apparent Mr of 132,000, suggesting noncovalent association as a tetramer. Studies with peptide p-nitroanilides indicated pronounced substrate preferences, with P1 arginine preferred to lysine. Benzoyl-L-Lys-Gly-Arg-p-nitroanilide was the best of the substrates screened. Inhibition by diisopropyl fluorophosphate and tosyllysine chloromethyl ketone indicated that the enzyme is a serine protease. Like the tryptases of human mast cells, mastocytoma tryptic protease was inhibited by NaCl, resistant to inactivation by alpha 1-proteinase inhibitor and plasma, and stabilized by heparin. Comparison of the N-terminal 24 residues of mastocytoma tryptase revealed 80% identity with the more limited sequence reported for human lung tryptase, and surprisingly, closer homology to serine proteases of digestion and clotting than to other leukocyte granule proteases sequenced to date, including mast cell chymase. The N-terminal isoleucine is the homolog of trypsinogen Ile-16 which becomes the new N-terminus upon cleavage of the activation peptide. Thus, the tryptase N-terminus is related to the catalytic domain of activated serine proteases, and lacks the N-terminal regulatory domains found in most clotting and complement serine proteases. These findings provide further evidence that tryptases are unique serine proteases and that they may be less closely related in evolution and function than are other leukocyte granule proteases described to date.

Our group developed two in vivo methods to study secretions from submucosal glands in exposed tracheal epithelium. (1) The exposed mucosal surface was coated with powdered tantalum; accumulated secretions produced elevations (hillocks) in the tantalum layer under which the duct openings were located. The rate of formation of the hillocks was observed through a dissecting microscope, and recorded by television on a video tape recorder. (2) Micropipets were used to collect timed samples from individual gland duct openings. With these techniques, the innervation of the submucosal glands and the autonomic regulation of their secretions were studied. We studied the control of ion movement across tracheal epithelium because active ion transport forms local osmotic gradients across epithelia which could regulate transepithelial water movement. We mounted pieces of the posterior wall of dog trachea in Ussing-type chambers and measured unidirectional fluxes of Cl- and Na+ under short-circuit conditions with 36Cl and 24Na. We found active transport of Cl- toward the lumen and Na+ toward the submucosa. With this technique we investigated the effect of parasympathomimetic drugs on ion movement. With a new in vitro method we studied output of 35S bound to sulfated mucins and movement of ions in cat trachea. We mounted pieces of anterior tracheal wall in Ussing-type chambers, added sodium 35S-sulfate to the submucosal side and monitored secretion of bound 35S in samples from the luminal side after dialysis. The unidirectional fluxes of Cl- and Na+ were measured with 36Cl and 22Na. With this method we examined the effect of alpha-adrenergic and beta-adrenergic agonists on mucin secretion and ion movement. Also with this preparation we studied the relationship between the permeability of the paracellular pathway to 14C-sucrose and the pattern of tight junction strands. Images FIGURE 1. FIGURE 4. FIGURE 6. FIGURE 7.

Neuropeptides such as substance P are implicated in inflammation mediated by sensory nerves (neurogenic inflammation), but the roles in disease of these peptides and the peptidases that degrade them are not understood. It is well established that inflammation is a prominent feature of several airway diseases, including viral infections, asthma, bronchitis, and cystic fibrosis. These diseases are characterized by cough, airway edema, and abnormal secretory and bronchoconstrictor responses, all of which can be elicited by substance P. The effects of substance P and other peptides that may be involved in inflammation are decreased by endogenous neutral endopeptidase (NEP; also called enkephalinase, EC 3.4.24.11), which is a peptidase that degrades substance P and other peptides. In the present study, we report that rats with histories of infections caused by common respiratory tract pathogens (parainfluenza virus type 1, rat corona-virus, and Mycoplasma pulmonis) not only have greater susceptibility to neurogenic inflammatory responses than do pathogen-free rats but also have a lower activity of NEP in the trachea. This reduction in NEP activity may cause the increased susceptibility to neurogenic inflammation by allowing higher concentrations of substance P to reach tachykinin receptors in the trachea. Thus decreased NEP activity may exacerbate some of the pathological responses in animals with respiratory tract infections.

Eosinophil major basic protein (MBP) is a granule-associated cytotoxic protein found in sputum and deposited on airway tissues of patients with acute asthma. We therefore studied the effect of human MBP on ion transport in dog tracheal epithelium. We mounted the posterior tracheal membranes of mongrel dogs in Ussing chambers and measured potential differences across the membranes and short-circuit current. Using 22Na+ and 36Cl- as tracers, we determined net ion movements. The addition of MBP (5 X 10(-6) M) to the mucosal, but not to the serosal, side of the membranes produced an increase in short-circuit current from 2.25 +/- 0.19 (mean +/- SE) to 2.78 +/- 0.23 muEq.cm-2h-1 (p less than 0.0001) and in net chloride secretion from 1.57 +/- 0.22 to 2.31 +/- 0.24 muEq.cm-2h-1 (p less than 0.01). There was no change in net sodium movement. Pretreatment with indomethacin (10(-5) M) attenuated, but did not abolish, the increase in short-circuit current. After exposure to MBP, prostaglandin E2 release into the serosal bathing solution increased from 10.0 +/- 4.2 to 17.0 +/- 6.9 ng.cm-2h-1 (p less than 0.05). The results of this study indicate that MBP simulates prostaglandin E2 production and chloride secretion by dog tracheal epithelium. Thus, eosinophils in the airways, through release of MBP, may affect mucociliary clearance by changing the volume and composition of respiratory tract fluid.

Mast cell tryptase is a secretory granule associated serine protease with trypsin-like specificity released extracellularly during mast cell degranulation. To determine the full primary structure of the catalytic domain and precursor forms of tryptase and to gain insight into its mode of activation, we cloned cDNAs coding for the complete amino acid sequence of dog mast cell tryptase and a second, possibly related, serine protease. Using RNA from dog mastocytoma cells, we constructed a cDNA library in lambda gt 10. Screening of the library with an oligonucleotide probe based on the N-terminal sequence of tryptase purified from the same cell source allowed us to isolate and sequence overlapping clones coding for dog mast cell tryptase. The tryptase sequence includes the essential residues of the catalytic triad and an aspartic acid at the base of the putative substrate binding pocket that confers P1 Arg and Lys specificity on tryptic serine proteases. The apparent N-terminal signal/activation peptide terminates in a glycine. A glycine in this position has not been observed previously in serine proteases and suggests a novel mode of activation. Additional screening of the library with a trypsinogen cDNA led to the isolation and sequencing of a full-length clone apparently coding for the complete sequence of a second tryptic serine protease (DMP) which is only 53.4% identical with the dog tryptase sequence but which contains an apparent signal/activation peptide also terminating in a glycine. Thus, the proteases encoded by these cloned cDNAs may share a common mode of activation from N-terminally extended precursors.

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, 113;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.

We studied whether antigen-induced airway hyperresponsiveness was associated with pulmonary inflammation in 11 anesthetized ragweed-sensitized dogs. Airway responsiveness to acetylcholine aerosol was determined before and at 2, 6, and 24 h after ragweed or sham aerosol challenge. Pulmonary inflammation was assessed by bronchoalveolar lavage (BAL) performed at either 2 or 6 h. Total pulmonary resistance increased 11-fold at 5 min after ragweed. Airway responsiveness was unchanged at 2 h but was increased 6.6-fold at 6 h in 8 of 11 dogs (P less than 0.001); hyperresponsiveness persisted from 4 days to 4 mo. Airway responsiveness was unchanged by aerosols of diluent. Neutrophils in BAL fluid increased approximately sixfold at 2 h (P less than 0.02) and at 6 h (P less than 0.02) after antigen challenge. There were fewer eosinophils in fluid recovered at 6 h after antigen compared with 2 h lavages (P less than 0.05). In three nonresponders, BAL showed no significant changes in neutrophils and eosinophils after antigen. Thus antigen-induced hyperresponsiveness is associated with the presence of pulmonary inflammation, presumably arising from the airways and involving both neutrophils and eosinophils.

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.

The enzyme responsible for 15-lipoxygenation of arachidonic acid was purified to homogeneity from human eosinophil-enriched leukocytes using a combination of ammonium sulfate precipitation, hydrophobic interaction chromatography, and high pressure liquid chromatography on hydroxyapatite and cation-exchange columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein revealed a single major band (apparent Mr 70,000). Amino acid sequence analysis yielded a single N-terminal sequence. Comparison of the N-terminal 15 residues reveals 71% sequence identity to the rabbit reticulocyte lipoxygenase and 36% sequence identity to the rat basophilic leukemia 5-lipoxygenase. In contrast, sequence identity to the soybean lipoxygenase-1 is not observed. These results demonstrate that human 15-lipoxygenase can be isolated from eosinophil-enriched leukocytes and is accessible for direct sequence analysis. Furthermore, we present initial evidence that the mammalian lipoxygenases constitute an homologous family of enzymes. The availability of homogeneous human 15-lipoxygenase will play a key role in elucidating other relationships in this family of enzymes.

A morphometric analysis was made of alterations in serous cell structure induced by adrenergic and cholinergic agonists. Ferret tracheal rings were exposed for 30 min in vitro to one of the following agonists: phenylephrine, terbutaline, or methacholine (all at 10(-5) M). Controls were incubated similarly in medium containing no drugs or medium containing both the agonist and an excess of the appropriate antagonist (phentolamine, propranolol or atropine, all at 10(-4) M). Electron microscopic observation and stereological analysis of the incubated samples revealed that the volume density of serous cell granules in controls (0.30 +/- 0.02, mean +/- SE, n = 4) was significantly reduced by phenylephrine (0.19 +/- 0.03, n = 4) and methacholine (0.17 +/- 0.01, n = 4), but not by terbutaline (0.27 +/- 0.04, n = 4). The presence of antagonists in the medium prevented the observed changes (phenylephrine/phentolamine: 0.29 +/- 0.03, n = 3 and methacholine/atropine: 0.33 +/- 0.06, n = 3). In addition, the volume density of intracellular vacuoles in controls (0.02 +/- 0.05, n = 4) was increased in response to methacholine stimulation (0.12 +/- 0.05, n = 4), but not in response to the other agonists. This effect was blocked by atropine (0.01 +/- 0.00, n = 3). We conclude that serous-cell granules are discharged by both alpha-adrenergic and cholinergic, but not beta-adrenergic stimulation. In addition, cholinergic stimulation evokes the formation of intracellular vacuoles, a possible indication of active ion and water transport.

The mechanisms of airflow limitation in bullous lung disease were examined by plotting maximal expiratory airflow and airway resistance against lung elastic recoil pressure. in 5 patients who had evidence of isolated bullae, the abnormalities in airway resistance and in expiratory airflow could be accounted for by loss of lung elastic recoil. in 2 additional patients, the abnormalities could not be accounted for completely by loss of lung elastic recoil, suggesting that intrinsic airway disease was also present; these 2 patients had clinically evident chronic bronchitis. in 2 of the patients, tantalum bronchography confirmed the physiologic studies. Postoperative studies in the one patient who underwent bullectomy demonstrated maintained physiologic improvement in lung elastic recoil and in airway resistance.

Powdered tantalum proved a superior tracheographic contrast material in 10 patients with symptomatic tracheal stenosis. It permitted assessment of degree of stenosis and extent of mucosal changes above and below the lesion. Only a small volume of the highly radiopaque material is required, and risk to the patient is minimal. The particles adhere to the mucosal surfaces and are not dislodged by cough or deep respiration. No deleterious effects on the respiratory function were noted, and the material appears biologically and clinically inert. Particles were cleared from the airways within six hours in all cases.

Inhalation of SO2 (4–6 ppm) for 10 min decreased airway conductance (increased airway resistance) in seven healthy subjects. Subcutaneous injection of atropine prevented this effect. In anesthetized, paralyzed, artifically ventilated cats, SO2 increased pulmonary resistance, whether delivered to the upper or to the lower airways. This was prevented by complete cold block of the cervical vagosympathetic nerves or by injecting atropine intravenously before the SO2 was inhaled. These results establish the reflex nature of bronchoconstriction during inhalation of SO2. Note: With the Technical Assistance of Earl H. White reflex bronchoconstriction; airway resistance Submitted on February 18, 1964

Equations for optimal frequencies of breathing at which mechanical work and mean inspiratory force are minimal and which assume dead space and resistance are constant ignore possible changes in airway caliber. We assumed an inverse relationship between resistance and dead space and plotted a series of curves relating mechanical work and mean inspiratory force to frequency of breathing for various dead spaces and corresponding resistances. As dead space increases, the levels of work and inspiratory force fall to minima, then rise; the frequencies at which work and force are minimal increase, then decrease. Adding a series resistance or dead space, changing the resistance/dead space relationship, decreasing lung compliance, or increasing alveolar ventilation do not alter the general shapes of the curves, but change the values of optimal dead space. The optimal dead spaces for minimal work (0.125 liter) and for minimal inspiratory force (0.14 liter) lie within the normal range. Nervously mediated airway tone in healthy subjects may represent optimal adjustment of airway caliber. dead space, optimal; bronchial caliber Submitted on October 26, 1962

The authors compared the effects of halothane and cyclopropane on total pulmonary resistance (RL) in dogs before (unstimulated) and during airway constriction produced by histamine or vagal nerve stimulation. In two dogs, direct measurements of airway diameter were obtained from bronchograms and com