The effect of tasuldine, a bronchosecretolytic agent, on mucus rheology and clearability and the interaction with acetylcholine in ferrets.

Tasuldine (Ts) is an orally active bronchosecretolytic agent shown to be clinically effective in human studies. Tasuldine decreases the sialomucin content of the mucus and, in animal studies, this modulation of the glycopeptide correlates with decreased mucus viscosity. The aim of this study was to evaluate the effect of tasuldine on mucus viscoelasticity and correlate the rheological changes to mucociliary and cough clearability. Tracheal mucus samples were collected from anaesthetized adult ferrets by a modification of the cytology brush technique. Mucus was collected prior to and following administration of either vehicle (normal saline) or Ts (10 mg/kg i.v.), and followed by acetylcholine (ACH) challenge (ca. 4 ml of 10(-2)M i.v., slow infusion). The analysis included magnetic microrheometry to measure the viscosity and elasticity of microlitre quantities of mucus. Mucociliary transportability (NFPTR) was measured by means of the frog palate assay and mucus collection rates (mg/min) were used as an indirect measure of secretion rate. The principal index of mucus rigidity, log G*, decreased with tasuldine infusion (P = 0.014) and further decreased with ACH (P = 0.002). In simple terms, the mucus became less rigid or more deformable with tasuldine administration, thus benefiting clearability based on predictions from model studies. The changes observed with acetylcholine alone were consistent with a classic secretagogue response--the outputting of a large volume of watery mucus. NFPTR increased with tasuldine treatment, and even further with acetylcholine; however, the combination of Ts and ACH resulted in a decrease in NFPTR close to baseline, which was likely due to the fact that the resulting mucus was too liquid for maximal mucociliary efficiency. The index of mucus flux (mg/min) was very much elevated with ACH compared with control; this was not the case with Ts. This indicates that tasuldine, despite improving the rheological properties of the mucus, did not stimulate hypersecretion, as was the case for acetylcholine. The changes in mucus rheology with infused tasuldine can be considered beneficial with respect to their effects on predicted mucociliary and cough clearability, supporting the clinical effectiveness of this type of mucolytic therapy in airway diseases such as chronic bronchitis. The study also illustrates the potential danger of overliquification of mucus.

Nicotine increases ciliary beat frequency by a direct effect on respiratory cilia.

We studied the direct effects of nicotine on the ciliary beat frequency (CBF) undisturbed by interference from mucus secretion by using epithelial strips from ferret tracheae which contain no goblet cells and, because the glands were left behind in the submucosa, no gland tissue either. Strips were studied in a chamber perfused with medium M-199 at 37 degrees C at a perfusion rate of 1.6 ml min-1 using a perfusion pump (Braun, Melsungen). CBF was determined photometrically, the signal being subjected to a fast Fourier transformation. We measured CBF continuously for 10 min (5 min with and 5 min without perfusion). Under baseline conditions without nicotine, it decreased from 23.4 +/- 0.8 to 22.2 +/- 0.5 Hz during perfusion and increased from 22.0 +/- 0.8 to 23.3 +/- 0.8 Hz during the subsequent period without perfusion. Nicotine increased CBF transiently, i.e., the effect was demonstrable only during perfusion, being strongest during the first 3 min of perfusion. At 10(-5) M, the increase in CBF was significant only during the 3rd minute, but at 10(-4) and 10(-3) M, CBF was elevated significantly throughout most of the perfusion period compared with control tissues perfused with medium M-199 only. Thus, at 2 min, CBF was 22.8 +/- 0.6 (mean +/- SE) in tissues perfused with medium M-199 only but was 24.3 +/- 0.8 Hz (NS, Student's unpaired t-test), 26.6 +/- 0.5 Hz (P = 0.001), and 26.8 +/- 1.2 Hz (P = 0.01) in tissues perfused with 10(-5), 10(-4), and 10(-3) M nicotine (dissolved in medium M-199), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Nicotine-induced airway smooth muscle contraction: neural mechanisms involving the airway epithelium. Functional and histologic studies in vitro.

To assess the mechanism of and the role of the epithelium in nicotine-induced bronchoconstriction in vitro, we performed a combined functional and histologic study. Functional study: We suspended tracheal strips or rings from 16 ferrets (1124 +/- 561 g, mean +/- SD) in organ baths. Alternate tracheal strips had their epithelium removed. Dose-response curves to acetylcholine (ACh) and nicotine were established for pairs of tissues with and without epithelium, each pair receiving only one dose of nicotine. Nicotine induced brief muscle contractions not exceeding 25% of the ACh-induced maximum. Contractions were blocked by hexamethonium and 10(-7) M atropine and were abolished or inhibited strongly by tetrodotoxin (TTX), suggesting the involvement of nicotinic neuronal and muscarinic smooth muscle receptors. Removal of the epithelium strongly inhibited contractions at concentrations of nicotine greater than 3 x 10(-5) M which completely removed any dose-response effect. ACh-induced contractions were unchanged, demonstrating smooth muscle integrity. We suggest that the removal of the epithelium attenuates nicotine-induced bronchoconstriction through the removal of nerves running in or close to the epithelium. Histologic study: In tracheae from 15 ferrets (8 male, 7 female), mean weight (+/- SD) 1288 (+/- 470) g, we examined 4 techniques of epithelium removal: (1) gentle scraping with a scalpel blade moved backwards (away from the cutting edge), (2) moving a Q-tip through the unopened tracheal tube without lateral pressure, and (3, 4) stroking the mucosa of opened tracheal segments with a Q-tip, exerting (3) light or (4) moderate pressure. All methods were equally (97%-100%) efficient in removing the epithelium but differed in the amount of damage caused to the basement membrane and/or submucosal tissue. Method (2) caused less damage to the basement membrane than the other methods but still removed almost one-third of it. The study showed that complete removal of the epithelium is at the expense of the submucosa and that a given result of "epithelium removal" is also attributable to removal of the neighboring subepithelial structures.

[Elastase-induced hyperfunction of submucous glands develops independent of elastase-induced emphysema]. / Die durch Elastase induzierte Uberfunktion submuköser Drüsen entwickelt sich unabhängig vom elastaseinduzierten Emphysem.

We instilled 2 mg of pancreatic elastase into the lungs, and insufflated a further 2 mg into the trachea of 6 male ferrets. Six control animals were given 0.9% NaCl (lungs) and glucose powder (trachea). This combined treatment was administered on days 1, 8 and 15. On these days, and again on days 29 and 39, we obtained chest x-rays. On the 39th day, we removed the lungs and measured the mean linear intercept. In tracheal sections, we measured goblet cell density, glandular structure and function by quantitative histology and autoradiography. Although, up to day 39, no emphysema developed, both structural and functional changes occurred in the glands: the volume density of the serous glands (S) decreased, that of the mucous glands (M) increased, the ratio S/M decreased from 2.0 to 1.5 (p = 0.05). The turnover of radioactive sulphur (indication of mucus synthesis) was significantly increased in animals treated with elastase (p = 0.001). Goblet cells increased from 0.9 +/- 0.4 to 3.4 +/- 1.8 cells per mm epithelium (p = 0.05). The results show that elastase induces structural and functional changes in submucosal glands and goblet cell hyperplasia, independently of emphysema.

[Effect of repeated nicotine administration on mucus secretion, bronchodilation and circulatory variables: inhibitory effect of autonomic antagonists]. / Wirkung wiederholter Nikotinapplikation auf Schleimsekretion, Bronchialweite und Kreislaufvariable: Hemmwirkung autonomer Antagonisten.

To investigate the mechanisms of action of nicotine we studied the effect of muscarinic and adrenergic receptor antagonists on nicotine-induced changes in the lungs and circulation. Nicotine increased mucus secretion from tracheal submucosal glands and caused bronchocon-striction. Cardiovascular effects were a bradycardia followed by tachycardia, hypertension and a biphasic increase in cardiac output. All of these effects were completely blocked by prior administration of hexamethonium. Propranolol and phentolamine prevented the increase in heart rate, and reduced the increase in blood pressure. Only atropine inhibited nicotine-induced hypersecretion and bronchocon-striction.

[Effect of recombinant gamma interferon on allergic skin reaction and bronchoconstriction]. / Wirkung von rekombinantem Interferon-gamma auf allergische Hautreaktion und Bronchokonstriktion.

Gamma-interferon (IFN-gamma) has immunosuppressive and immunostimulatory effects. Net results in clinical disease are difficult to predict. We studied possible anti-allergic effects of IFN-gamma in a double blind study in 30 patients with bronchial asthma. We performed quantitative skin prick and bronchial allergen provocation tests using each patient's main allergen prior to and after 20 injections of either recombinant IFN-gamma (100 micrograms each) or placebo, measuring skin wheal area, specific airway resistance (SRaw) and the maximal expiratory flow at 50% of vital capacity (MEF50). IFN-gamma reduced skin wheal area significantly but had no effect on allergic bronchoconstriction.

[Stimulation of mucous glands of the respiratory tract by neuropeptides: comparison of exogenous administration and endogenous liberation]. / Stimulierung von Schleimdrüsen der Atemwege durch Neuropeptide: Vergleich exogener Zufuhr und endogener Freisetzung.

Exogenous substance P is a powerful stimulant of tracheal gland secretion but the contribution of endogenous neuropeptides to neurogenic gland secretion is unknown. Using the Ussing chamber technique, we measured the secretion of radiolabeled macromolecules from submucosal glands in the ferret. Neurokinins caused gland secretion through a neurokinin-1 (NK-1) receptor. Gland secretion caused by electric field stimulation of postganglionic nerve endings was not inhibited by a substance P antagonist and was not augmented by agents known to prevent neurokinin degradation in the tissue. We conclude that the release of endogenous neurokinins plays no rôle in neurally evoked gland secretion. This is despite the fact that endogenous neurokinins have been shown to be involved in vagally induced airway smooth muscle contraction and increased capillary permeability in the same species.

[Pharmacologic modification of mediator liberation and the effect of mediators]. / Pharmakologische Beeinflussbarkeit der Mediatorfreisetzung und der Mediatorwirkung.

Blocking specific steps in the chain of events leading to disease is essential for elucidating pathogenesis. If antagonism to a given receptor does not modify the disease the receptor plays no role in the disease. In this review we describe specific points in the chain of events leading to respiratory disease at which pharmacologic intervention is possible and we describe results of such intervention. This includes interventions not presently available therapeutically. We distinguish three groups of interventions: interventions interfering with pre-receptor mechanism, those interacting with pharmacologic receptors or with proteins coupling receptor activation to cellular events and, finally, interventions affecting postreceptor mechanisms, e.g., second messenger systems including ion channels. Of the many interventions possible early pre-receptor interventions are most desirable (preventive measures), but are often not available. Interventions at the receptor level, although available and in most cases specific for a given receptor are of limited effectiveness because multiple receptor involvement predominates in respiratory disease. Interventions at the post-receptor level have been most successful thus far presumably because post receptor mechanisms (e.g. second messenger systems) are shared by many receptors, therefore interventions at this level are largely independent of receptor(s) involved and have been most effective clinically.

Stimulation of submucosal glands by nicotine applied locally to the airway mucosa.

Chronic bronchitis is characterized by hypersecretion of mucus and is caused by cigarette smoking. We investigated the effect of nicotine on mucus secretion from tracheal submucosal glands, applying nicotine to the airway mucosa in vitro. We anesthetized 50 ferrets with pentobarbital (66 mg/kg), excised their tracheae and mounted tracheal segments in Ussing chambers. We added 50 microCi Na(2)35SO4 (35S) to the submucosal side and determined nondialyzable 35S in medium collected from the luminal side at 15 min intervals. Nicotine was a powerful stimulant of mucus secretion with threshold effects at 10(-5) M and peak effects at 3 x 10(-4) M. Percentage increases were the same for males and females, but absolute increases in mucus secretion were significantly larger in males than in females. Luminal nicotine was more effective than submucosal nicotine, especially when nicotine bitartrate was used (increase above baseline, 150 +/- 54 vs 22 +/- 12 cpm/15 min, 10(-4) M, n = 4, bitartrate). Effects of luminal nicotine sulfate were larger than those of luminal nicotine bitartrate (303 +/- 88 vs 120 +/- 38 cpm/15 min, P less than 0.05, n = 6, 10(-4) M). Two applications of nicotine 1.5 h apart had similar effects up to 3 x 10(-5) M. At higher concentrations the second response was significantly weaker than the first (tachyphylaxis). Secretory effects of nicotine were prevented completely by atropine and were reduced significantly by hexamethonium.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of systemic nicotine on mucus secretion from tracheal submucosal glands and on cardiovascular, pulmonary, and hematologic variables.

To investigate effects of systemic nicotine on mucus secretion from tracheal submucosal glands we anesthetized 13 ferrets (5 male, 8 female; average weight 1138 +/- 469 g, mean +/- SD) with pentobarbital (initial dose, 62 +/- 26, total dose, 90 +/- 26 mg/kg), cannulated the jugular vein for i.v. application of infusions and drugs and cannulated the femoral artery for determination of blood pressure, heart rate, blood cells, and blood gases. We opened the thorax, cannulated the trachea 1 cm above the carina and ventilated the lungs through the lower airways with a Harvard respirator. We placed an electromagnetic flow probe around the ascending aorta for measurement of cardiac output. We measured transpulmonary pressure as tracheal pressure with a strain gauge transducer. We created an isolated segment of trachea between the larynx and the tracheal cannula. We perfused the segment with medium M-199 containing 30 microCi/ml Na(2)35SO4 (35S) and we injected 100 microCi 35S intravenously. After 90 min we drained the radioactive solution from the luminal side and replaced it with nonradioactive medium which we collected at 5-min intervals for determination of nondialyzable radioactivity. At 25 min we injected nicotine sulfate (5 x 10(-7)-10(-5) M/kg) and continued to collect the perfusate every 5 min. Systemic nicotine had profound effects on circulatory and ventilatory variables and on gland secretion. Initial hypertension was followed by bradycardia and a fall in blood pressure and cardiac output.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ozone on breathing in dogs: vagal and nonvagal mechanisms.

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.

Ion transport across cat and ferret tracheal epithelia.

Sheets of trachea from ferret and cat were mounted in Ussing chambers and continuously short circuited. Under resting conditions, in both the cat and ferret there was little or no Cl secretion, and Na absorption accounted for most of the short-circuit current (Isc). Ouabain (10(-4) M, serosal bath) reduced Isc to zero in 30-60 min. This decline was matched by a decrease in net Na absorption. Amiloride (10(-4) M, luminal bath) caused a significant decrease in Isc and conductance (G) in both species. Bumetanide (10(-4) M, serosal bath) had negligible effects on Isc and G. In both species, isoproterenol increased Isc by stimulating Cl secretion. Methacholine induced equal amounts of Na and Cl secretion, with little change in Isc. In the cat, prostaglandins E2 and F2 alpha and bradykinin increased Isc, responses which were abolished in Cl-free medium. In open-circuited cat tissues, Na flux from the serosal to mucosal side was measured simultaneously with the secretion of nondialyzable 35S. Prostaglandins E1, E2, and F2 alpha, histamine, bradykinin, methacholine and isoproterenol all increased both Na and 35S-mucin secretion.

Role of the parasympathetic nervous system and of cholinergic mechanisms in bronchial hyperreactivity.

The parasympathetic nervous system of the respiratory tract is involved in the control of airway calibre in three ways: through afferent nerve pathways (pulmonary reflexes); through efferent nerve pathways (reflexes, interaction between efferent vagus and mediators or modulating transmitter substances) and through cholinergic muscarinic receptors and postreceptor mechanisms in the target organ. To what extent do these mechanisms contribute to airway hyperreactivity? Pulmonary reflexes: Reflex bronchoconstriction has been established in divided lung experiments for histamine and in experiments involving an isolated segment of trachea for SO2. A reflex pathway is the most likely explanation for the heightened reactivity induced by aerosols of prostaglandin E2 and by maximal respiratory manoeuvers. Reflex bronchoconstriction is mediated through rapidly adapting ("irritant") receptors and through C-fibre endings. The influence of C-fibre endings is greater than hitherto suspected and many effects ascribed to irritant receptors are probably due to stimulation of C-fibre endings which outnumber myelinated fibres 3-4 to 1. From studies on the control of respiration (which reflect more directly the state of sensory receptors in the airways than studies of airway calibre) it appears that the activity of C-fibre endings increases during ozone-induced hyperreactivity. This could explain the increased bronchial reactivity seen in this condition. Interaction: Aerosols of serotonin cause bronchoconstriction when the vagus nerve is intact but have little effect during vagal block. This is an effect neither on afferent receptors nor on the end organ, but on the efferent nerve pathway. Interaction effects of this type ("cholinergic facilitation") are frequent and may be more important quantitatively than reflex effects. From data on serotonin and by analogy to other systems it appears that preganglionic and ganglionic sites are important points of interaction. Parasympathetic ganglia are located in the airway wall. Several transmitter substances have been identified in airway ganglia and in autonomic nerves, sensory and motor. Thus there seem to be convergent inputs capable of modulating transmission through the ganglia. An altered balance of converging ganglionic inputs may cause hyperreactivity. Receptors in airway smooth muscle may not be a homogeneous population. Muscarinic receptors are being classified into subgroups and the existence of at least 3 subtypes: M-1, M-2 and M-3 has been postulated. Their distribution depends on the tissue studied. They differ in agonist affinity messenger systems.(ABSTRACT TRUNCATED AT 400 WORDS)