We used scanning electron microscopy to count the number of mucous gland openings in the tracheae and lower portion of the larynges of the rat, guinea pig, hamster, mouse and rabbit. Cells of the airway surface epithelium were removed by protease digestion better to visualise the gland openings. The distribution of glands was further studied by conventional histology and by PAS\Alcian blue staining of whole mounts. In all rodent species, gland openings in the larynx occurred with a frequency of 1-2 per mm#. Mice had no gland openings in their tracheae, and hamsters, only a handful. Rat tracheae contained 126p42 gland openings (p..; n l 6) at a frequency of " 0n6 per mm# at the top of the trachea and " 0n15 per mm# at the bottom. Guinea pig tracheae contained 153p90 gland openings (p..; n l 5), with 54 % being in the top 40 % of the trachea. In both rat and guinea pig, tracheal glands were found in the ventral aspect between the cartilaginous rings, and were absent from the dorsal membranous portion. Gland openings in most species were simple circles of " 50 µm diameter. However, glands in the rat trachea generally opened obliquely into shallow (" 20 µm deep) oval troughs (" 150i75 µm), which had their long axes oriented from head to tail. In the rabbit, there was no evidence of tracheal or laryngeal glands histologically. However, the tracheal and laryngeal surfaces contained numerous pits (" 30 µm diameter) distributed evenly over and between cartilages at a frequency of " 4 per mm#. These may correspond to the ' nests ' of goblet cells described by others.

The luminal surface of airways is lined by a thin film of airway surface liquid (ASL). Physiological regulation of the depth of ASL has not been reported previously. In this paper, we have used low-temperature scanning electron microscopy of rapidly frozen specimens of bovine tracheal epithelium to demonstrate alterations in the depth of ASL in response to the cholinergic agonist methacholine. We first established that methacholine selectively stimulated airway glands, with maximal secretion at ∼2 min and a return to baseline within ∼5 min. A 2-min exposure to methacholine increased the depth of ASL from 23 to 78 μm. Thereafter, depth decreased linearly with time, reaching 32 μm at 30 min. The initial increase in depth was blocked by bumetanide, an inhibitor of active chloride secretion, whereas the slow decline back to baseline was inhibited by amiloride, a blocker of active sodium absorption. We conclude that the methacholine-induced changes in ASL depth reflect transient gland secretion followed by liquid absorption across the surface epithelium.

R Re eg gu ul la at ti io on n o of f d de ep pt th h a an nd d c co om mp po os si it ti io on n o of f a ai ir rw wa ay y s su ur rf fa ac ce e l li iq qu ui id dThe airways are lined with a film of fluid, which is 5-20 µm deep in healthy individuals. This fluid is believed to consist of two phases [1]. A watery fluid of low viscosity, the "periciliary sol" surrounds the cilia. Above this lies a viscous mucous gel. The cilia are able to beat in the sol. Their tips contact the underside of the mucous blanket and propel it towards the mouth. This system of "mucociliary clearance" serves to remove particles trapped in the mucous gel. The concept of two layers of airway surface liquid (ASL) has been supported by a variety of microscopy studies [2][3][4][5] (fig. 1).Factors influencing the liquid content of sol and gel have been identified but their relative contributions are imperfectly understood. Water will move into the airway lumen in response to active Cl -secretion across the epithelia of the tracheal surface and of submucosal glands. Active absorption of Na + across the surface epithelium serves to remove liquid. These active ion transport processes move liquid by generating local osmotic gradients across the epithelium [6]. Hydrostatic pressure gradients across the epithelium, and differences in osmotic pressure between the media bathing the epithelium's two surfaces, will also influence the net movement of liquid into or out of the lumen. Evaporation is an important factor in the upper airways. Finally, forces of surface tension generated by the closely packed cilia SERIES "AIRWAY MUCUS" Edited by P.K. JefferyNumber 6 in this Series may serve to hold liquid in the airway lumen [7]. These factors are considered in turn below. Additionally, we describe recent experiments in which we have used lowtemperature scanning electron microscope (LT-SEM) to determine the depth and composition of the airway surface liquid (ASL). Gland secretionThe adult human trachea has about 10 submucosal gland openings per mm 2 of airway surface [8]. It is unknown how the structure and numbers of glands change down the airways, except that bronchi have glands, whereas bronchioles do not [9]. In each gland, a ciliated duct leads from the airway surface to an expanded collecting duct into which empty ~10 secretory tubules, each with multiple branches ending in closed acini [10]. The acini are lined by serous cells, the tubules by mucous cells [11]. It is believed that the serous cells secrete fluid which serves to flush out mucins released by the more proximal mucous cells [12]. Consistent with this hypothesis, gland serous cells contain much the highest levels of the cystic fibrosis transmembrane conductance regulator of any cell type in the airways [13] Respir J 1997; 10: 1914-1917