Paul S. Richardson

Heterogeneity of airways mucus: variations in the amounts and glycoforms of the major oligomeric mucins MUC5AC and MUC5B.

Respiratory mucus contains a mixture of gel-forming mucins but the functional significance of these different mucin species is unknown. To help gain a better understanding of mucus in airways we therefore need to ascertain the concentration of each of the gel-forming mucins within respiratory secretions. Thus the aim of this study was to determine the amounts of specific gel-forming mucins directly from solubilized secretions of the airways and purified mucin preparations. We investigated the feasibility of using direct-binding ELISA employing mucin-specific antisera but were unable to obtain reliable data owing to interference with the immobilization of the mucins on the assay surface by 6 M urea and high levels of non-mucin proteins. We therefore developed an alternative approach based on quantitative Western blotting after agarose-gel electrophoresis, which was not subject to these problems. Here we demonstrate that this procedure provides reliable and reproducible data and have employed it to determine the amounts of the MUC2, MUC5AC and MUC5B mucins in saline-induced sputa from healthy airways and spontaneous sputa from asthmatic airways. Additionally we have used this procedure to analyse these glycoproteins in mucin preparations purified from cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) mucus. Our findings indicate that MUC5AC and MUC5B are the major oligomeric mucins and that airways mucus contains variable amounts of these glycoproteins. By contrast, the MUC2 mucin comprised, at most, only 2.5% of the weight of the gel-forming mucins, indicating that MUC2 is a minor component in sputum. Finally, we show that the amounts and glycosylated variants of the MUC5AC and MUC5B mucins can be altered significantly in diseased airways with, for instance, an increase in the low-charge form of the MUC5B mucin in CF and COPD mucus.

The Composition of Tracheal Mucus and the Nervous Control of Its Secretion in the Cat

A new method for measuring the output of mucus proteins (specifically the sulphated glycoproteins) from the trachea of anaesthetized cats is described. The method has been used to measure the effects of nervous and pharmacological stimuli on mucus output and the mucus collected has been fractionated and analysed chemically. There is a resting output of mucus which does not depend on autonomic nerves. We have shown that sympathetic nerve stimulation and sympathomimeticamines increase tracheal mucus output. These effects were prevented by β-adrenergic blocking agents but not by α-adrenergic blockade. Sympathetic efferent fibres to tracheal mucus glands run through the stellate ganglia, then some of the fibres pass up into the lower part of the cervical sympathetic nerves before looping back into the chest and passing rostrally again to the trachea. We have confirmed that parasympathetic nerve (vagal) stimulation increases mucus protein output and shown that the strength of this effect is about the same as that of sympathetic nerve stimulation. Atropine blocked this effect. Pilocarpine, a parasympathomimetic agent, greatly increased mucus protein output. Chemical analysis of the secretions showed that they contained two groups of glycoproteins which could be separated by electrophoresis. Only the slower-moving of these was sulphated. Gel filtration on Sephadex G-200 and ion-exchange chromatography on DEAE-cellulose DE-52 showed that the 35S-sulphated material was heterogeneous in both molecular size and charge density. The principal monosaccharides in tracheal mucus glycoproteins are galactose, N-acetylglucosamine, N-acetylgalactosamine, fucose and sialic acid. Mannose was not detected. Three cell types contributed to the glycoprotein secretion of the cat trachea, mucous and serous cells of the submucosal glands and goblet cells of the overlying epithelium. The histochemical investigations suggest that most mucous and goblet cells produced a sulphated mucin; the results for the serous cells are ambiguous. Autoradiography confirms all three cell types produced sulphated secretions which were labelled when 35SO4 was given intrasegmentally. The tracheal mucosa showed only minor degrees of damage in a small proportion of cases.

The control of mucin secretion into the lumen of the cat trachea by α- and β-adrenoceptors, and their relative involvement during sympathetic nerve stimulation

Abstract We have tested the effects of phenylephrine, dobutamine and salbutamol, α-, β1- and β2-adrenoceptor agonists respectively, on the output of radiolabelled mucins into the cat trachea in situ. Phenylephrine significantly increased mucin output, an effect inhibited by the α-adrenoceptor antagonists, thymoxamine or prazosin, but not by propranolol. Dobutamine increased the output of 35S-labelled mucins greatly and had a smaller effect on 3H-labelled mucins. Propranolol blocked these effects but thymoxamine did not. At high doses atenolol, a β1-adrenoceptor antagonist, inhibited dobutamines effect on 35S-labelled mucins. Salbutamol caused a small increase in mucin output and propranolol blocked this increase. Electrical stimulation of the sympathetic nerve supply to the trachea increased mucin output. Propranolol inhibited this effect; thymoxamine did not. We conclude that both α- and β-adrenoceptors increase mucus secretion into the cat trachea but that only the β-adrenoceptors respond to sympathetic nerve stimulation.

Mucus-glycoproteins (mucins) of the cat trachea: characterisation and control of secretion.

Glycoproteins produced by the tracheae of anaesthetized cats were radiolabelled biosynthetically by a pulse administration of Na2 35SO4 and [3H]glucose into the tracheal lumen. Subsequently, radiolabelled secretions were washed from the tracheal lumen. Repeated doses of pilocarpine and then ammonia vapour were given to stimulate secretion. Pilocarpine-stimulated glycoproteins, which came mainly from the submucosal glands, were particularly enriched with 35S. Ammonia-stimulated secretions, which probably came mostly from the microvillous border of the surface epithelium, contained mainly 3H radioactivity but little 35S. Two negatively-charged glycoproteins of different molecular size were identified in the secretions: the larger component was excluded on Sepharose CL-4B and it had a higher 3H 35S ratio than the smaller component which was retarded on Sepharose CL-4B. The relative amount of the smaller component decreased progressively with repeated pilocarpine stimulation and it was not detected in secretions induced by ammonia. Pilocarpine stimulation caused little alteration in carbohydrate composition of the secreted glycoproteins. In response to ammonia, glycoproteins were secreted with a high sialic acid content but quantitatively they represented a small amount of material compared with that induced by pilocarpine. These findings suggest that tracheal glycoproteins from different epithelial-cell sources have distinctive chemical compositions and that their secretions may be independently regulated. The 35S-rich high-molecular-weight glycoproteins from the submucosal glands were of the mucin-type but those derived from the microvillus border may represent a different class of airway glycoproteins from typical epithelial mucins.

Respiratory Mucus: Structure, Metabolism and Control of Secretion

Mucus has at least three important roles in the protection of the airways: (i) It is essential for the transport of dust, debris, irritants and bacteria from the lungs. Two transport mechanisms clean the airways. Ciliated cells line all the conducting airways (i.e. those concerned with distributing air to the gas exchanging parts of the lung rather than the process of gas exchange itself) and these beat continuously to propel mucus from the smaller to the larger airways and eventually out of the lung altogether. In the absence of mucus or something with equivalent physical properties, cilia are powerless to move dust etc. (King et al, 1974). The other cleaning mechanism is cough which is only called upon when the burden of irritation or debris threatens to overwhelm the cilia. As with mucociliary transport, mucus is essential for effective coughing. A dry cough fails to remove dust from the lungs (Yeates et al, 1975). (ii) Mucus also has a role in diluting irritants which enter the airway and so it renders them less harmful. (iii) There is growing evidence that mucus has antibacterial and antiviral properties. It contains immunoglobulins, principally secretory IgA (Kaltreider, 1976)., lactoferrin which chelates iron necessary for the growth of some bacteria (Masson & Heremans 1966) and lyzozyme which destroys some bacteria (Lorenz et al, 1957).

Mucus Hypersecretion and Its Role in the Airway Obstruction of Asthma and Chronic Obstructive Pulmonary Disease

Several distinct processes may narrow the airway lumen in the diseased lung: smooth muscle contraction, airway scarring and remodelling, encroachment of submucosal tissue swollen with oedema or engorged blood vessels, collapse of the airway walls under a pressure gradient and accumulation of mucus. Only in the last of these, the subject of this chapter, is the obstruction situated within the airway lumen itself; the remainder result from processes in or even outside the airway walls. It is rare to find that a single mechanism entirely accounts for airway obstruction in any patient, though one may predominate in a particular phase of his or her illness.

Decreasing the particle size of aerosols of local anaesthetic by heating

We used a cascade impactor and radioactive labelling to determine the distribution of particle size in aerosols from several commercially available nebulizers, and the effect of heating on the size distribution. Mass median diameter (MMD) of the unheated aerosols ranged from 1.4 to 4.8 μm and this was reduced by heating to a range of 06 to 1.9 μm. Bupivacaine (0.5%) can be given as an aerosol after heating with minimal side-effects from anaesthesia of the upper respiratory tract, since the smaller, concentrated, particles are deposited deeper in the lung. This may have clinical applications.