Paul W. Kent

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 composition and biosynthesis of the glycoproteins and glycolipids of the rabbit small-intestinal brush border.

1. The glycoprotein and glycolipid composition of isolated rabbit small-intestinal brush borders has been studied. 2. The total glycoprotein fraction contains an average 95 microgram carbohydrate per mg protein, composed of mannose, galactose, fucose, N-acetylglucosamine and N-acetylgalactosamine. Glucose is also present but sialic acid is absent. 3. The isolated glycolipids include ceramide lactoside, ceramide trihexoside and two N-acetylglucosamine-containing glycolipids. Sialic acid containing glycolipid (gangliosides) is present only in trace quantities. 4. The biosynthesis of the brush border-bound glycoproteins and glycolipids has been studied following intraperitoneal injection with D-[1-14C]glucosamine and isolation of the brush borders at intervals between 3 and 24 h. 5. The total glycoprotein fraction labels maximally 7.5 h after injection and subsequently exhibits an exponential loss of radioactivity with a half-life of 11.2 h. The labelling kinetics of one of the glucosamine-containing glycolipids is similar to that of the glycoproteins in that it labels maximally between 7.5 and 12 h, but the second glucosamine-containing glycolipid labels later at approximately 18 h. These results indicate that the glycoproteins and glycolipids are actively synthesized and degraded within the mature small intestinal enterocyte and that individual glycolipids turn over independently.

Factors influencing the viscous properties of chicken tracheal mucins

1. Reduced viscosities, in water, of different types of mucin, such as fibrillar, gelatinous and soluble phase, separated from chicken tracheal secretions were measured. 2. H-bond breaking agents caused a significant decrease in the reduced viscosity of these mucins, but thiol-reagents alone did not have any effect. 3. Papain and Pronase did not cause any decrease in the reduced viscosity of these mucins. Neuraminidase decreased the reduced viscosity of soluble phase mucin by 50% by removing about 30% of its N-acetylneuraminic acid but had no effect on fibrillar and gelatinous mucins. Sulphatase neither removed any sulphate ester groups nor decreased the reduced viscosity. Due to some nonspecific intermolecular interaction, mixtures of mucins and enzymes or ovalbumin exhibited elevated reduced viscosities. 4. Ionic strength of the solutions appeared to decrease the reduced viscosity of these mucins. Increasing concentrations of Ca2+ in solutions of ionic strength of approx. 0.1 caused significant decrease in the reduced viscosity, but had no such effect in solutions of ionic strength of more than 0.1. 5. N-Acetylneuraminic acid and sulphate ester residues were 46.6 +/- 0.2, 43.4 +/- 0.6, 27.9 +/- 3.3 mg/g and 66.0 +/- 2.0, 34.2 +/- 3.3, 2.5 +/- 0.8 mg/g for fibrillar, gelatinous and soluble phase mucins, respectively. There appeared to be a good correlation between viscosity and N-acetylneuraminic acid contents among mucins of low reduced viscosities and between viscosity and sulphate ester residues among mucins of high reduced viscosities.

Role of directional Ca2+ effect on reduced viscosities of mucus secretions from chicken trachea in vitro

Reduced viscosities of fibrillar and gelatinous type mucins produced in response to high submucosal Ca2+ and low luminal Ca2+ effects were significantly higher than those of corresponding types of normal mucins. The increased reduced viscosity of experimental mucin samples was due to their aggregation with unique low molecular weight (mr 325,000 and 46,200) sulphate-rich components. The Ca2+ appeared to exert two opposing effects on viscosity properties of mucins; whereas Ca2+-dependent complexes between different types of mucins appeared to be a selective phenomenon between sulphate-rich mucins and components. Ester sulphate residue content rather than N-acetylneuraminic acid residue content of these mucins and low molecular weight components showed a very good correlation with their reduced viscosity and Ca2+-binding values.

Directional Ca2+ effect on stimulation of secretion of common mucins and unique sulphate-rich components from chicken trachea in vitro

High submucosal Ca2+ (3.6-18 mM) significantly increased the secretion of a common high molecular weight fibrillar mucin (approx. Mr is greater than 2.10(6)) and also elicited the secretion of an additional low molecular weight component (approx. Mr 325,000). Low luminal Ca2+ (0.018 mM) also significantly increased the secretion of a common high molecular weight gelatinous mucin (approx. Mr is greater than 2.10(6)) and elicited the secretion of an additional low molecular weight component (approx. Mr 46,200). The additional low molecular weight components were more heavily sulphated (6.7 and 4.2%) than common high molecular weight mucins (2.1 and 1%). The low molecular weight components and high molecular weight mucins were secreted as aggregates which could be dissociated by EGTA. The low molecular weight components and high molecular weight mucins were different in the number of their glycoprotein constituents and in the ion-exchange chromatographic profiles and the carbohydrate and ester sulphate residue content of their acidic glycoproteins.