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CONTROL OF MUCUS
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SECRETION AND ION TRANSPORT IN AIRWAYS Jay A. Nadel, Brian Davis, and Roger
J.
Phipps
of Medicine and Physiology, University of California, San Francisco, California
Cardiovascular Research Institute and Departments
94143
INTRODUCTION
Mucus and water combine in a complex way to form the respiratory tract secretions. Electron micrographs (33) indicate that vesicles in the mucous and serous cells of the submucosal glands and in the goblet and serous cells of the airway epithelia discharge granules of mucus into the gland ducts and into the airway lumen, respectively. These granules are hydrated, in some unknown way, and form a coating of secretion over the ciliated epithelium of the airways. This secretion consists of an upper gel (38, 78) and a lower, more fluid sol in which the cilia can beat freely and sweep the gel, with its trapped inhaled particles, up the airway to be swallowed. Excellent reviews of the comparative anatomy of airways (12), chemistry of mucus (44), rheology of airway mucus secretion (36, 65) mechanisms of ciliary beating (63), and mucociliary clearance from the lungs (73) and the nose (61) give detailed information about these aspects of the structure and function of airways. In this review we discuss recent advances in the regula tion of mucoprotein secretion and the possible role of ion transport in the regulation of water secretion into the airway lumen. New techniques allow the functions of the submucosal glands to be separated from those of the surface epith..dial cells. Therefore, as far as possible, we discuss the physi ology of these subunits independently. Although the role of ion transport in the movement of fluid across most other mammalian epithelia has been studied extensively, the investigation of ion transport in airway epithelia was initiated only recently and promises to provide important new informa tion about the physiology of the airways. ,
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MUCUS SECRETION Studies of tracheobronchial mucus secretion are hampered by the small volume of secretions normally produced, by the relative inaccessibility of the sources of these secretions, and by the limited sensitivity of techniques available to analyze the small amounts of glycoproteins collected. Many studies have involved the collection of sputum, but because sputum is contaminated with both saliva and nasal mucus its study gives little infor mation about the normal control of airway secretions. Since mucus is transported toward the mouth, methods have been employed to collect the total secretions at a given level of the airway (e.g. in the trachea) (I, 55). However, these techniques are limited because it is not known where the collected secretions originate, and because these methods are likely to stim ulate mucus secretion by local irritation and reflex mechanisms. Neverthe less, the use of tracheal segments in vivo (20, 23), explants of trachea and bronchi (9, 68), isolated pieces of trachea mounted in chambers (57), and micropipettes to obtain secretions from specific structures (e�g. submucosal gland duct openings) (51) have provided useful information about the vari ous sources of airway mucus.
Submucosal Glands These are found in the trachea and bronchi, but not in bronchioles. They are numerous in several species, including humans (70), cats (20, 23), and pigs (7); infrequent or nonexistent in some species, including rabbits and guinea pigs (45); and nonexistent in other species, including geese (59) and chickens (72). Each gland is comprised of four distinct regions: (a) a short, funnel-shaped ciliated duct that is a continuation of the surface epithelium; (b) a nonciliated collecting duct; (c) mucous tubules, lined with mucous secretory cells, opening into the collecting duct; and (d) serous tubules, lined with serous secretory cells, opening into mucous tubules (49) (Figure 1). Both mucous and serous cells produce glycoproteins; in addition, the serous cells probably also produce other proteins (44). Myoepithelial cells closely related to the secretory cells (8, 48) may aid the passage of secretion along the tubules by contracting and squeezing the cells. The evidence for parasympathetic efferent innervation of the submucosal glands is strong. Electron-microscopic studies in humans show nerve fibers that contain agranular vesicles near mucous and serous cells, which sug gests the presence of postganglionic cholinergic efferent nerve endings (8, 48). Anatomic studies using a specific acetylcholinesterase stain confirm the presence of a cholinergic innervation of submucosal glands (19, 40, 74). Stimulation of the efferent parasympathetic nerve supply to the airways increases both the volume of the secretions (20, 50) and the output of
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�---Serous Ce"
Submucosal Gland Duct Afferent Nerve
Figure 1
Diagram of secretory cells in airway. Serous (
•
) and mucous (
0)
secretions from
the appropriate cells in the submucosal gland combine with water to form the submucosal gland secretion, which is discharged via the gland duct onto the airway luminal surface. This secretion mixes with the mucous and serous secretions from the epithelial goblet and serous cells to coat the epithelial surface with an upper gel and a lower more fiuid sol in which the cilia beat and propel the gel toward the mouth. The apical surfaces of some cells are covered by microvilli whose function is unknown. Golgi apparatus ('�) of the secretory epithelial cells, and nuclei
(�,), endoplasmic reticulum (�), and mitochondria (j®) in the other
surface cells are shown. Endings of cholinergic afferent nerves in the lateral intercellular spaces close to the junctions between epithelial cells send impulses to the central nervous system and refiexly stimulate secretion from the serous and mucous cells of the submucosal glands via cholinergic efferent nerves.
radiolabelled glycoprotein (23). These effects are cholinergic since they are mimicked by cholinergic agonists, and both nerve and drug effects can be blocked by muscarinic antagonists. There is no direct evidence for a para sympathetic nervous effect in human airway submucosal glands, but cholin ergic agonists stimulate glycoprotein secretion in humans in vitro (9, 68), as they do in other species (13). The evidence that these cholinergic effects are, at least in part, due to glandular secretions derives from two sources. First, histologic studies following vagal stimulation show a decrease in glycoprotein primarily in the submucosal glands (both mucous and serous cells) (20, 23). Second, using a new technique that we have developed to visualize secretions from gland duct openings in vivo, we showed that stimulation of the efferent vagus nerves causes localized fluid accumulation above the gland ducts (52).
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Stimulation of cough receptors in the trachea and bronchi with ammonia
increases tracheal mucus output reflexly via parasympathetic efferent path ways, but stimulation of irritant receptors in peripheral airways does not (58) It is postulated that stimulation of the cough reflex has the following .
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reflex components: (0) expulsive expiration; (b) narrowing of large bronchi and trachea, perhaps increasing shear rates in airways during cough; and (c) mucus secretion, thus increasing the mucus barrier and perhaps also increasing the effectiveness of removal of the foreign irritant during cough (58). It is interesting that cough receptors, submucosal glands, and sites of airway compression during cough have a similar distribution! Stimulation
of other pulmonary receptors has no apparent effect on tracheal mucus secretion (58). In cats, irritation of the nose, pharynx, or larynx also reflexly increases mucus secretion, partly via parasympathetic efferent nerves (58). Chemicals given to the stomach increase the output of respiratory tract fluid, but the efferent pathways are not known (55). Direct sympathetic nervous regulation of submucosal glands is less cer tain. The presence of dense-cored vesicles in nerve fibers near human sub mucosal gland cells is suggestive of an efferent adrenergic innervation (48), but the evidence concerning the existence of these fibers is conflicting (8).
No adrenergic effect on airway mucus secretion could be shown in several species (11,13, 59,68),but some of these findings may be due to inadequate knowledge of anatomy or to species variations. Thus, the lack of an effect of electrical stimulation of the cervical sympathetic nerves on tracheal mucus in rabbits and guinea pigs could be due to the fact that the main sympathetic nerve supply to the trachea usually derives from the stellate ganglia; or it could be due to the fact that these species contain little "mucus-secreting tissue" (11). In cats, electrical stimulation of the stellate ganglia increases tracheal mucus secretion via a �-adrenergic action (23).
Irritation of the nose, pharynx, or larynx also increases tracheal mucus secretion in cats, partly via sympathetic pathways (58). Similarly, adrener gic agonists stimulate mucus secretion in cats in vivo via a tJ-adrenergic action (23),while in vitro studies suggest an additional a.-adrenergic action (B. Davis, R. J. Phipps, J. A. Nadel, unpublished). It may be that specific
adrenergic agonists stimulate receptors in different cells, some responsible for volume secretion and others for the secretion of concentrated muco proteins. This could explain the finding that a specific tJz-agonist produced secretions with normal viscosity, whereas other tJ-agonists produced secre tions with increased viscosity (47). Could the higher death rate in asthmatic patients associated with the use of high doses of nonspeciftc � -agonists have been caused by the production of viscid mucus, with subsequent plugging of airways (56, 66)7
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Epithelial Cells Two epithelial cell types in the airway are sources of glycoproteins: goblet cells and epithelial serous cells. The goblet cells resemble the goblet cells of the intestine and the mucous cells of the airway submucosal glands. They are found mainly in the trachea and bronchi, decrease in number toward the periphery, and are infrequent in bronchioles. Goblet cells are numerous in several species, including humans (76), cats (20, 23), pigs (6), and geese (59), but are sparse in other species, such as rabbits, guinea pigs (45) and healthy rats (32). Epithelial serous cells resemble serous cells of the airway submucosal glands. Their distribution is similar to that of goblet cells, and they have so far been found in the airway of humans (33), cats (30), and geese (59). A third secretory cell type, the Clara cell, is found in several species, mainly in the bronchioles (12). Whether they secrete lipid or protein is unclear. They may have the capacity to produce either lipid or glycoprotein depend ing upon environmental conditions: Airway irritation with smoke or S02 induces transitions of Clara cells to goblet cells and stimulates the produc tion of glycoprotein by these cells (33). A sulfated glycoprotein has been shown to line the luminal surface of airway ciliated cells, including the cilia, in humans and dogs (67), and in cats and geese (30, 56). Its chemical composition is different from that of submucosal gland glycoproteins; it is probably secreted from epithelial cells (22, 24). Evidence for an innervation of epithelial secretory cells is conflicting, and there is probably much species variation. Specific acetylcholinesterase stain ing shows cholinergic efferent fibers in the epithelia of pigs (40): Specific immunofluorescent staining shows adrenergic efferent fibers in the epithelia of pigs (40) and rats (79). Efferent nerve profiles have been observed in the airway epithelia of rats (31) and geese (59): In both these species fibers containing agranular vesicles and fibers containing dense-cored vesicles were found. Some of these fibers in both species were observed near secre tory cells. The presence of a cholinergic innervation of epithelial secretory cells in geese (a species with only epithelial secretory cells and no sub mucosal glands) is confirmed by the finding that tracheal mucus secretion increased following parasympathetic nerve stimulation (59).
Effects of Irritants Acute administration of irritant agents to the airways, including organic vapors (10), ammonia, cigarette smoke, and CS solution (O-chlorobenzili dine malononitrile) (62), increases airway mucus secretion. Inhaled am monia vapor and cigarette smoke stimulate secretion from both epithelial
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secretory cells and submucosal glands, and cause release of a surface muco substance (30, 56). These irritants act partly through reflex neural pathways (perhaps involving stimulation of cough receptors) and partly through a direct effect: The reflex stimulation probably causes secretion from the submucosal glands, and the direct stimulation probably causes secretion from the mucosal cells (56, 62). The mechanism of this direct effect is not clear, but it may involve prostaglandins as intermediaries (62). Chronic administration of S02 (32) or cigarette smoke (34) to the airways increases airway mucus secretion and produces goblet cell hyperplasia, transforma tion of epithelial cells, and enlargement of submucosal glands.
Effects of Drugs Various pharmacologic mediators affect mucus secretion. Histamine, a putative mediator in asthma, stimulated mucus secretion in cats and geese in vivo (62), but had no effect in vitro either in dogs (13) or in humans (68). The discrepancies could be due to species differences. Local application of prostaglandin (PG)El stimulated mucus secretion in various airways of cats (62) and rats (29); also, PGF2a when given locally stimulated mucus secre tion in cats (62), and when inhaled as an aerosol induced sputum production in healthy humans (37). PGF2a stimulates cough receptors, and since part of the effect of this drug on bronchomotor tone is mediated by cholinergic efferent pathways (75), the secretory effects of PGF2a could similarly be mediated partly via cholinergic efferent pathways, although a direct effect cannot be excluded. Tissue inflammation causes local PG release, and such release could account for the increase in mucus secretion found in inflam matory airway diseases. Basic polypeptides (including kallidin and sub stance P) increased tracheal glycoprotein secretion in dogs in vitro, while a kalladin antagonist (hexadrimethrine) decreased it (5). Local application of the anesthetic lidocaine to the tracheas of cats or geese not only blocked the responses to known secretogogues, but also stimulated glycoprotein secretion directly (56, 59). The mechanisms of these actions are unknown but may involve the displacement of calcium ions from extra- and intracel lular binding sites (56). ACTIVE ION TRANSPORT
Since water plays such an important role in the formation of the respiratory tract secretions, we hypothesized that the regulation of water movement into the airway lumen might be important in detennining the physical properties of the mucus layer and the rate of its movement up the airway by the cilia (51). Therefore, we sought methods for the study of water movement across the airway epithelium. It is generally accepted (28) that
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water crosses absorptive (14,1 7,64) and secretory (18, 69) epithelia as a result of local osmotic gradients created by active ion transport. To investi gate ionic fluxes in airway epithelia,we used Ussing's short-circuit current method (71) and pieces of epithelium from the posterior membranous part of dog tracheas. We found a net flux of CI- toward the lumen and a smaller net flux of Na+ toward the submucosa; these net fluxes accounted for the measured short-circuit current (53). Therefore, CI- and Na+ are actively transported across tracheal epithelium and may be the only actively trans ported ions. Our findings have been confirmed subsequently in dogs (2), and suggestive evidence has been provided that CI- and Na+ are transported similarly in rabbits (46) and cats (57) and that Cl- is actively transported toward the lumen in rats (39). Evidence presented for active K+ transport in rabbits (46) is unconvincing. Submucosal gland cells and surface epithelial cells may both be involved in active ion transport. However,since rabbits have no tracheal submucosal glands but their tracheas actively transport CI- and Na+,the sites of active ion movement in this species must be in the surface epithelial cells. Active secretion of CI- may be a property of all epithelial tissues derived from the primitive foregut,since it occurs in the amphibian (27) and mammalian (35) stomach, the mammalian esophagus (60), and the amphibian (25) and mammalian (54) lung. Furosemide reduced net CI- movement toward the lumen when added to the submucosal side,but not when added to the luminal side of the tracheal epithelium (16). Ouabain was bound to more sites on the submucosal mem brane than on the luminal membrane of the tracheal epithelium (77); this binding is presumably to Na+-K+ATPase (4,77),the sodium pump. These findings suggest that active CI- and active Na+ transport occur at the submucosal membrane. CI- and Na+ transport are interdependent,since net CI- movement is greatly reduced by replacement of Na+ in the bathing solution [(2,43); J. H. Widdicombe,personal communication], and by add ing ouabain, an inhibitor of sodium pumps, to the submucosal bathing solution (2,77). In one experimental model for the link between Na+ and CI- movement, CI- enters the cells by a Na+-linked system across the submucosal membranes,the transmembrane Na+ gradient providing the energy for active accumulation of Cl- by the cells. The Na+ entering with the CI- is pumped back to the submucosa by basolaterally placed Na+ pumps,while the CI- diffuses down an electrochemical gradient across the luminal surfaces of the cells. The energy for CI- secretion is thus provided by the transmembrane Na+ gradient and ultimately therefore by the Na+ pump (J. H. Widdicombe,personal communication). The electrical resistances and the spontaneous electrical potential differ ences across epithelia correlate well with physical parameters such as
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permeability to electrolytes, hydraulic conductivity, and the ability of epith elia to maintain an osmotic gradient (21). Airway epithelia of different species have electrical resistances about 300 ohm cm2 and a range of sponta neous electrical potential differences between 20 and 60 mV. This classifies them as "tight junction" epithelia and implies that, relative to "leaky junc tion" epithelia (e.g. rat jejunum), they are less permeable to electrolytes, are poor conductors of water, and can maintain large transepithelial osmotic gradients. Two patterns of electrical potential differences across the luminal and submucosal membranes of rabbit tracheal epithelial cells have been ob served from intracellular recordings. One group found some cells with the inside negative to both the luminal surface and the submucosal surface, and other cells with the inside positive to the luminal surface and negative to the submucosal surface (26). Another group found only cells with the inside negative to both the luminal surface and the submucosal surface, and they attributed the potential differences to the following differences in the per meabilities of each cell membrane to electrolytes: In the luminal membrane the permeability to CI- > S042- > Na+ > K+, and in the submucosal membrane the permeability to K+ > Na+ > CI- > SOi- (46). Further studies of the intracellular distribution of ions and the associated electrical potentials should help to determine the role of active ion transport in transmembrane water movement. We studied the effects of drugs that mimic the actions of the autonomic nervous system, and the effects of mediators on active ion transport across pieces of tracheal epithelium mounted in Ussing chambers. Acetylcholine increased net movement of Cl- and Na+ toward the canine tracheal lumen. The electrical resistance of the tissue was unchanged, and most of the increase of ion movement was electrically neutral. The effect was prevented by small concentrations of atropine (41). Terbutaline, a specific f1z-adrener gic agonist, increased net movement of Cl-, but not of Na+, toward the tracheal lumen in dogs (15) and cats (57). The electrical potential difference increased despite a fall in electrical resistance of the tissue. Propranolol prevented the effects in dogs. The effects of epinephrine (3) were similar to those of terbutaline. Also, terbutaline given intravenously to dogs increased the potential difference between a fluid-filled tracheal segment in situ and the paratracheal fascia (B. Davis, M. G. Marin, J. A. Nadel, unpublished). Phenylephrine, a specific a-adrenergic agonist, increased net movement of CI- and Na+ toward the tracheal lumen in cats (D. Davis, R. J. Phipps, J. A. Nadel, unpublished). The major effect was stimulation of Na+ secretion into the lumen, which was measured under open-circuit conditions. Since phenylephrine had no effect on the electrical properties of the posterior membranous part of tracheal epithelium in dogs (B. Davis, M. G. Marin,
MUCUS SECRETION AND ION TRANSPORT IN AIRWAYS
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A. Nadel, unpublished), the marked effect on the anterior portion of
tracheal epithelium in cats may indicate either a species difference or an effect on submucosal glands, which are abundant in the anterior part of cat tracheas and sparse in the posterior membranous part of dog tracheas. Histamine increased net movement of CI- and Na+ toward the tracheal lumen. The increase in total ion transport was dose-related and could be prevented by an HI-antagonist, but not by an Hz-antagonist (42). The electrical potential difference was increased despite a small decrease in
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tissue resistance. Thus, the effects of autonomic agonists and mediators on the output of respiratory tract secretions could be due in part to their effects on ion transport.
SUMMARY The output of secretions from the airway submucosal glands is regulated by vagal efferent nerves. Stimulation of cough receptors increases mucus output reflexly via the vagus nerves. Adrenergic agonists increase sub mucosal gland secretions in some species, which indicates that adrenergic receptors are present in these cells. However, evidence for adrenergic ner vous pathways to the glands is limited. Irritants and drugs stimulate secre tion from epithelial cells by direct effects. There is also evidence that the secretion of epithelial cells can be stimulated by parasympathetic nervous pathways in birds but not in mammals. Active ion transport of Cl- toward the lumen and of Na+ toward the submucosa results in net ion movement toward the airway lumen in unstimulated tracheal epithelia. Drugs and mediators increase the net movement of ions toward the lumen. No agents have yet been found that increase net ion movement toward the submucosa. The link between ion transport and water secretion in airway epithelia, although speculative, seems likely in view of the evidence from other epith elia. Since airway epithelium is a "tight junction" epithelium, modification of the tight junctions may alter the transepithelial movement of water and ions. We suggest that the depth and consistency of the periciliary layer of airway secretions determine the ability of the cilia to propel the muco protein gel and thereby modify mucociliary transport. To achieve this, secretion of mucus must be controlled separately from the secretion of water. Studies are needed to determine which of the speCialized functions of the epithelial cells interact to regulate the clearance of secretions from the airway. Is the sol maintained by secretion and reabsorption of fluid across the epithelium? Does the sol move with the gel by ciliary action or does it remain stationary? Do changes in the epithelial tight junctions influence net water movement and thus indirectly alter the depth of the sol layer? To answer these questions, techniques are needed to study subunits
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of the airway, including isolated surface cells and submucosal glands; and sensitive methods are required to analyze the very small samples of secre tions for glycoprotein and electrolyte content. Intracellular measurements of electrolyte concentrations and electrical potentials may help to elucidate the mechanisms of transepithelial ion and water movement. The control system for the production and removal of respiratory tract secretions may be altered in disease. For instance, chronic stimulation of cough receptors causes reflex secretion and may be the cause of the hyper plasia of submucosal glands and of the abnormal secretions that occur in chronic bronchitis and asthma (50, 58). The abnormally viscid mucus in cystic fibrosis may be due to a defect in CI- transport, which provides too little water for both the gel and sol layers. These speculations are intended to identify areas for further research, which hopefully will reduce the mor bidity and mortality in these common lung diseases. ACKNOWLEDGMENTS
We thank Ms. Linda Morehead and Mrs. Beth Cost for typing and prepara tion of the manuscript, and Mr. Robert W. Surface for medical illustration. During the preparation of this review, Dr. Davis was supported by Young Investigator Research Award HL-21150 from the National Heart, Lung and Blood Institute. Some of the reported studies were supported by grants from the U.S. Public Health Service: Program Project HL-06285 and Pul monary SCOR Grant HL-19156, and in part by a grant from the Cystic Fibrosis Foundation. Literature Cited 1. Adams, G. K., Aharonson, E. F., Rea sor, M . J., Proctor, D. F. 1976. Collec tion of normal canine tracheobronchial secretions. J. Appl. Physiol. 40:247-49 2. Al-Bazzaz, F., Al-Awqati, Q. 1977. Characteristics of ion transport in ca nine tracheal epithelia. Fed. Proc. 36(3):479 (Abstr.) 3. AI-Bazzaz, F. J., Khan, A., Cheng, E. 1977. Stimulation of chloride secretion across canine tracheal epithelia by theo phylline and epinephrine. Clin. Res. 25:414A (Abstr.) 4. AI-Bazzaz, F. J., Westenfelder, C., Ear nest, W., Kurtzman, N. 1977. Charac terization of canine tracheal epithelium Na-K-ATPase. C/in. Res. 25:413A (Abstr.) 5. Baker, A. P., Hillegass, L. M., Holden, D. A., Smith, W. J. 1977. Effect ofkalli din, substance P, and other basic poly peptides on the production of respira-
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MUCUS SECRETION AND ION TRANSPORT IN AIRWAYS 11. Boyd, E. M., Jackson, S., Ronan, M. 1943. The effect of sympathomimetic amines upon the output of respiratory tract fluid in rabbits. Am. J. Physiol 138:565-68 12. Breeze, R. G., Wheeldon, E. B. 1977. The cells of the pulmonary airways. Am. Rev. Resp. Dis. 116:705-77 13. Chakrin, L. W., Baker, A. P., Christian, P., Wardell, J. R. 1973. Effect of cholin ergic stimulation on the release of mac romolecules by canine trachea in vitro. Am. Rev. Resp. Dis. 108:69-76 14. Curran, P. F., MacIntosh, J. R. 1962. A model system for biological water trans port. Nature 193:347-48 15. Davis, B., Marin, M. G., Nadel, J. A. 1975. ,8-adrenergic receptor in canine tracheal epithelium. Am. Rev. Resp. Dis. 111:947 (Abstr.) 16. Davis, B., Marin, M. G., Ueki, I., Na del, J. A. 1977. Effect of furosemide on chloride ion transport and electrical properties of canine tracheal epi thelium. Clin. Res. 25:132 (Abstr.) 17. Diamond, J. M., Bossert, W. H. 1967. Standin� gradient osmotic flow: A mecharusm for coupling of water and solute transport in epithelia. J. Gen. Physiol 50:2061-83 18. Diamond, J. M., Bossert, W. H. 1968. Functional consequences of ultrastruc tural geometry in "backwards" fluid transporting epithelia. J. Cell BioI. 37:694-702 19. EI-Bermani, Al-W. I., Grant, M. 1975. Acetylcholinesterase-positive nerves of the rhesus monkey bronchial tree. Tho rax 30:162-70 20. Florey, H., Carleton, H. M., Wells, A. Q. 1932. Mucus secretion in the tra chea. Br. J. Exp. Pathol 13:269-84 21. Fromter, E., Diamond, J. 1972. Route of passive ion permeation in epithelia. Nature New Biol. 235:9-13 22. Gallagher, J. T., Hall, R. L., Jeffery, P. K., Phipps, R. J., Richardson, P. S. 1977. The nature and origin of tracheal secretions released in response to pilo carpine and ammonia. J. PhysioL Lon don 275:36-37P (Abstr.) 23. Gallagher, J. T., Kent, P. W., Pass atore, M., Phipps, R. J., Richardson, P. S. 1975. The composition of tracheal mucus, and the nervous control of its secretion in the cat. Proc. R. Soc. Lon don Ser. B. 192:49-76 24. Gallagher, J. T., Kent, P. W., Phipps, R. J., Richardson, P. S. 1977. Influence of pilocarpine and ammonia vapor on the secretion and structure of cat tra cheal mucins: differentiation of goblet
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effect of tobacco smoke with or without the anti-inflammatory agent phenyl methyloxadiazole. Br. J. Exp. Pathol. 54:229-39 Kitahara, S. 1967. Active transport of Na+ and CI- by in vitro nonsecreting cat gastric mucosa. Am. J. Physiol 213:819-23 Litt, M. 1973. Basic concepts of mucus rheology. Bull Physio-Pathol Resp. 9:33-46 Lopez-Vidriero, M. T., Reid, L. 1978. Bronchial mucus in health and disease. Br. Med. Bull. 34:63-74 Lucas, M. A., Douglas, L. C. 1934. Principles underlying ciliary activity in
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NADEL, DAVIS & PHIPPS the respiratory tract. II. A comparison of nasal clearance in man, monkey and other mammals . Arch. OtolaryngoL 20:518-41 Mangos, J. A. 1976. Fluxes of elec trolytes and water in rat trachea. Cystic Fibrosis 15:17 (Abstr.) Mann, S. P. 1971. The innervation of mammalian bronchial smooth muscle: the localization of catecholamines and cholinesterases. Histochem. J. 3:319-31 Marin, M. G., Davis, B., Nadel, J. A. 1976. Effect of acetylcholine on Cl- and Na+ fluxes across dog tracheal epi thelium in vitro. Am. J. Physiol. 231: 1546-49 Marin, M. G., Davis, B., Nadel, J. A. 1977. Effect of histamine on electrical and ion transport properties of tracheal epithelium. Am. J. PhysioL 42:735-38 Marin, M. G., Zaremba, M. M. 1978. Influence of Na+ on stimulation of net Cl- fiux by acetylcholine and terbutaline in dog tracheal epithelium. Fed. Proc. 37(3):514 (Abstr.) Masson, P. L., Heremans, J. F. 1973. Sputum proteins. In Sputum, Funda mentals and Clinical Pathology, ed. M. Duifano, pp. 412-75. Springfield, 111.: Charles C. Thomas. 632 pp. Mawdsley-Thomas, L. E., Healey, P., Barry, D. H. 1971. Experimental bron chitis in animals due to sulphur dioxide and cigarette smoke. An automated quantitative study. In Inhaled Particles III Vols. 1, 2, ed. W. H. Walton, pp. 509-26. London: Unwin. 1090 pp. Melon, J. 1968. Activite secretoire de la muqueuse nasale. Acta Otorhinolaryn goL Belg. 22:11-244 Melville, G. N., Horstmann, G., Iravani, 1. 1976. Adrenergic com pounds and the respiratory tract. A physiological and electron-microscopi cal s tud y. Respiration 33:261-69 Meyrick, B., Reid, L. 1970: Ultrastruc ture of cells in the human bronchial sub mucosal glands. J. Anat. 107:281-99 Meyrick, B., Sturgess, J. M., Reid, L. 1969. A reconstruction of the duct sys tem and secretory tubules of the human bronchial submucosal gland. Thorax 24:729-36 Nadel, J. A. 1977. Autonomic control of airway smooth muscle and airway secretions. Am. Rev. Resp. Dis. 115: (Suppl.) 117-26 Nadel, J. A., Davis, B. 1977. Auto nomic regulation of mucus secretion and ion transport in airways. In Asthma IL ed. K. F. Austen, L. M. Lichten-
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stein, pp. 197-210. New York: Aca demic. 414 pp. Nadel, J. A., Davis, B. 1978. Regulation of Na and Cl transport and mucous gland secretion in airway epithelium. See Ref. 30, pp. 133-47 Olver, R. E., Davis, B., Marin, M. G., Nadel, J. A. 1975. Active transport of Na+ and Cl- across the canine tracheal epithelium in vitro. Am. Rev. Resp. Dis. 112:811-15 Olver, R. E., Strang, L. B. 1974. Ion fluxes across the pulmonary epithelium and the secretion of lung liquid in the foetal lamb. J. PhysioL London 241: 327-57 Perry, W. F., Boyd, E. M. 1941. A method for studying expectorant action in animals by direct measurement of the output of respiratory tract fluids . J. Pharmacol. Exp. Ther. 73:65-67 PhiP s, R. J. 1977. The control 0/ tra chea mucin secretion. Ph D Thesis, London Univ., England. 341 pp. Phipps, R., Davis, B., Nadel, J. A. 1978. Effect of terbutaline on mucin secretion in cat airway using a new in vitro method. Fed. Proc. 37(3):221 (Abstr.) Phipps, R. J., Richardson, P. S. 1976. The effects of irritation at various levels of the airway upon tracheal mucus secretion in the cat. J. PhysioL London 261:563-81 Phipps, R. J., Richardson, P. S., Cor field, A., Gallagher, J. T., Jeffery, P. K., Kent, P. W., Passatore, M. 1977. A physiological, biochemical and histo logical study of goose tracheal mucin and its secretion. Philos. Trans. R. Soc. London Ser. B. 279:513-43 Powell, D. W., Morris, S. M., Boyd, D. D. 1975. Water and electrolyte t rans port by rabbit esophagus. Am. J. PhysioL 229:438-43 Proctor, D. F. 1977. The upper airway, I. Nasal physiology and defense of the lungs. Am. Rev. Resp. Dis. 115:97-129 Richardson, P. S., Phipps, R. J., Balfre, K., Hall, R. 1978. The roles of media tors, irritants and allergens in causing mucin secretion from the trachea. See Ref. 30, pp. 111-31 Satir, P. 1974. The present status of the sliding microtubule model of ciliary motion. In Cilia and Flagel/a, ed. M. A. Sleigh, pp. 131-42. London and New York: Academic SOO pp. Schultz, S. G. 1977. The role of para ce1lular pathways in isotonic fluid trans port. Yale J. BioL Med. 50:99-113 Silberberg, A., Meyer, F. A., Gilboa, A., Gelman, R. A. 1977. Function and
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properties of epithelial mucus. See Ref. 24 pp. 171-80 Speizer, F. E., Doll, R., Heaf, P., Strang, L. B. 1968. Investigation into use of drugs preceding death in asthma. Br. Med. J. 1:335-39 Spicer, S. S., Chakrin, L. W., Wardell, J. R., Kendrick, W. 1971. Histochemis try of mucosubstances in the canine and human respiratory tract. Lab. Invest. 25:483-90 Sturgess, I., Reid, L. 1972. An organ culture study of the effect of drugs on the secretory activity of the human bronchial submucosal gland. Clin. Sci. 43:533-43 Swanson, C. H. 1977. Isotonic water transport in secretory epithelia. Yale J. BioL Med. 50:153-63 Thurlbeck, W. M., Benjamin, B., Reid, L. 1961. Development and distribution
of mucous glands in the foetal human trachea. Br. J. Dis. Chest 55:54-64
71. Ussing, H. H., Zerahn, K. 1951. Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiol. Scand.
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72. Walsh, C., McLelland, I. 1974. The ul trastructure of the avian extrapulmo-
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89:412-22 73. Wanner, A. 1977. Clinical aspects of mucociliary transport. Am. Rev. Resp.
Dis. 116:73-125
74. Wardell, I. R. Ir., Chakrin, L. W., Payne, B. J. 1970. The canine tracheal pouch: a model for use in respiratory mucus research. Am. Rev. Resp. Dis. 101:741-54 75. Wasserman, M. A. 1975. Bronchopul monary responses to prostaglandin Fz", histamine and acetylcholine in the dog. Eur. J. Pharmacol. 32:146-55 76. Watson, J. R. L., Brinkman, G. L. 1964. Electron microscopy of the epi thelial cells of normal and bronchitic human bronchus. Am. Rev. Resp. Dis.
90:851-66
77. Widdicombe, J. R., Yee, J. Y., Nadel, J. A. 1978. Site of Na-pumps in dog tra cheal epithelium. Fed. Proc. 37(3):221 (Abstr.) 78. Yoneda, K. 1976. Mucous blanket of rat bronchus: an ultrastructural study. Am. Rev. Resp. Dis. 114:837-42 79. Zussman, W. V. 1966. Fluorescent lo calization of catecholamine stores in the rat lung. Anal. Rec. 156:19-30
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CONTROL OF MUCUS
+1225
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SECRETION AND ION TRANSPORT IN AIRWAYS Jay A. Nadel, Brian Davis, and Roger
J.
Phipps
of Medicine and Physiology, University of California, San Francisco, California
Cardiovascular Research Institute and Departments
94143
INTRODUCTION
Mucus and water combine in a complex way to form the respiratory tract secretions. Electron micrographs (33) indicate that vesicles in the mucous and serous cells of the submucosal glands and in the goblet and serous cells of the airway epithelia discharge granules of mucus into the gland ducts and into the airway lumen, respectively. These granules are hydrated, in some unknown way, and form a coating of secretion over the ciliated epithelium of the airways. This secretion consists of an upper gel (38, 78) and a lower, more fluid sol in which the cilia can beat freely and sweep the gel, with its trapped inhaled particles, up the airway to be swallowed. Excellent reviews of the comparative anatomy of airways (12), chemistry of mucus (44), rheology of airway mucus secretion (36, 65) mechanisms of ciliary beating (63), and mucociliary clearance from the lungs (73) and the nose (61) give detailed information about these aspects of the structure and function of airways. In this review we discuss recent advances in the regula tion of mucoprotein secretion and the possible role of ion transport in the regulation of water secretion into the airway lumen. New techniques allow the functions of the submucosal glands to be separated from those of the surface epith..dial cells. Therefore, as far as possible, we discuss the physi ology of these subunits independently. Although the role of ion transport in the movement of fluid across most other mammalian epithelia has been studied extensively, the investigation of ion transport in airway epithelia was initiated only recently and promises to provide important new informa tion about the physiology of the airways. ,
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MUCUS SECRETION Studies of tracheobronchial mucus secretion are hampered by the small volume of secretions normally produced, by the relative inaccessibility of the sources of these secretions, and by the limited sensitivity of techniques available to analyze the small amounts of glycoproteins collected. Many studies have involved the collection of sputum, but because sputum is contaminated with both saliva and nasal mucus its study gives little infor mation about the normal control of airway secretions. Since mucus is transported toward the mouth, methods have been employed to collect the total secretions at a given level of the airway (e.g. in the trachea) (I, 55). However, these techniques are limited because it is not known where the collected secretions originate, and because these methods are likely to stim ulate mucus secretion by local irritation and reflex mechanisms. Neverthe less, the use of tracheal segments in vivo (20, 23), explants of trachea and bronchi (9, 68), isolated pieces of trachea mounted in chambers (57), and micropipettes to obtain secretions from specific structures (e�g. submucosal gland duct openings) (51) have provided useful information about the vari ous sources of airway mucus.
Submucosal Glands These are found in the trachea and bronchi, but not in bronchioles. They are numerous in several species, including humans (70), cats (20, 23), and pigs (7); infrequent or nonexistent in some species, including rabbits and guinea pigs (45); and nonexistent in other species, including geese (59) and chickens (72). Each gland is comprised of four distinct regions: (a) a short, funnel-shaped ciliated duct that is a continuation of the surface epithelium; (b) a nonciliated collecting duct; (c) mucous tubules, lined with mucous secretory cells, opening into the collecting duct; and (d) serous tubules, lined with serous secretory cells, opening into mucous tubules (49) (Figure 1). Both mucous and serous cells produce glycoproteins; in addition, the serous cells probably also produce other proteins (44). Myoepithelial cells closely related to the secretory cells (8, 48) may aid the passage of secretion along the tubules by contracting and squeezing the cells. The evidence for parasympathetic efferent innervation of the submucosal glands is strong. Electron-microscopic studies in humans show nerve fibers that contain agranular vesicles near mucous and serous cells, which sug gests the presence of postganglionic cholinergic efferent nerve endings (8, 48). Anatomic studies using a specific acetylcholinesterase stain confirm the presence of a cholinergic innervation of submucosal glands (19, 40, 74). Stimulation of the efferent parasympathetic nerve supply to the airways increases both the volume of the secretions (20, 50) and the output of
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�---Serous Ce"
Submucosal Gland Duct Afferent Nerve
Figure 1
Diagram of secretory cells in airway. Serous (
•
) and mucous (
0)
secretions from
the appropriate cells in the submucosal gland combine with water to form the submucosal gland secretion, which is discharged via the gland duct onto the airway luminal surface. This secretion mixes with the mucous and serous secretions from the epithelial goblet and serous cells to coat the epithelial surface with an upper gel and a lower more fiuid sol in which the cilia beat and propel the gel toward the mouth. The apical surfaces of some cells are covered by microvilli whose function is unknown. Golgi apparatus ('�) of the secretory epithelial cells, and nuclei
(�,), endoplasmic reticulum (�), and mitochondria (j®) in the other
surface cells are shown. Endings of cholinergic afferent nerves in the lateral intercellular spaces close to the junctions between epithelial cells send impulses to the central nervous system and refiexly stimulate secretion from the serous and mucous cells of the submucosal glands via cholinergic efferent nerves.
radiolabelled glycoprotein (23). These effects are cholinergic since they are mimicked by cholinergic agonists, and both nerve and drug effects can be blocked by muscarinic antagonists. There is no direct evidence for a para sympathetic nervous effect in human airway submucosal glands, but cholin ergic agonists stimulate glycoprotein secretion in humans in vitro (9, 68), as they do in other species (13). The evidence that these cholinergic effects are, at least in part, due to glandular secretions derives from two sources. First, histologic studies following vagal stimulation show a decrease in glycoprotein primarily in the submucosal glands (both mucous and serous cells) (20, 23). Second, using a new technique that we have developed to visualize secretions from gland duct openings in vivo, we showed that stimulation of the efferent vagus nerves causes localized fluid accumulation above the gland ducts (52).
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Stimulation of cough receptors in the trachea and bronchi with ammonia
increases tracheal mucus output reflexly via parasympathetic efferent path ways, but stimulation of irritant receptors in peripheral airways does not (58) It is postulated that stimulation of the cough reflex has the following .
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reflex components: (0) expulsive expiration; (b) narrowing of large bronchi and trachea, perhaps increasing shear rates in airways during cough; and (c) mucus secretion, thus increasing the mucus barrier and perhaps also increasing the effectiveness of removal of the foreign irritant during cough (58). It is interesting that cough receptors, submucosal glands, and sites of airway compression during cough have a similar distribution! Stimulation
of other pulmonary receptors has no apparent effect on tracheal mucus secretion (58). In cats, irritation of the nose, pharynx, or larynx also reflexly increases mucus secretion, partly via parasympathetic efferent nerves (58). Chemicals given to the stomach increase the output of respiratory tract fluid, but the efferent pathways are not known (55). Direct sympathetic nervous regulation of submucosal glands is less cer tain. The presence of dense-cored vesicles in nerve fibers near human sub mucosal gland cells is suggestive of an efferent adrenergic innervation (48), but the evidence concerning the existence of these fibers is conflicting (8).
No adrenergic effect on airway mucus secretion could be shown in several species (11,13, 59,68),but some of these findings may be due to inadequate knowledge of anatomy or to species variations. Thus, the lack of an effect of electrical stimulation of the cervical sympathetic nerves on tracheal mucus in rabbits and guinea pigs could be due to the fact that the main sympathetic nerve supply to the trachea usually derives from the stellate ganglia; or it could be due to the fact that these species contain little "mucus-secreting tissue" (11). In cats, electrical stimulation of the stellate ganglia increases tracheal mucus secretion via a �-adrenergic action (23).
Irritation of the nose, pharynx, or larynx also increases tracheal mucus secretion in cats, partly via sympathetic pathways (58). Similarly, adrener gic agonists stimulate mucus secretion in cats in vivo via a tJ-adrenergic action (23),while in vitro studies suggest an additional a.-adrenergic action (B. Davis, R. J. Phipps, J. A. Nadel, unpublished). It may be that specific
adrenergic agonists stimulate receptors in different cells, some responsible for volume secretion and others for the secretion of concentrated muco proteins. This could explain the finding that a specific tJz-agonist produced secretions with normal viscosity, whereas other tJ-agonists produced secre tions with increased viscosity (47). Could the higher death rate in asthmatic patients associated with the use of high doses of nonspeciftc � -agonists have been caused by the production of viscid mucus, with subsequent plugging of airways (56, 66)7
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Epithelial Cells Two epithelial cell types in the airway are sources of glycoproteins: goblet cells and epithelial serous cells. The goblet cells resemble the goblet cells of the intestine and the mucous cells of the airway submucosal glands. They are found mainly in the trachea and bronchi, decrease in number toward the periphery, and are infrequent in bronchioles. Goblet cells are numerous in several species, including humans (76), cats (20, 23), pigs (6), and geese (59), but are sparse in other species, such as rabbits, guinea pigs (45) and healthy rats (32). Epithelial serous cells resemble serous cells of the airway submucosal glands. Their distribution is similar to that of goblet cells, and they have so far been found in the airway of humans (33), cats (30), and geese (59). A third secretory cell type, the Clara cell, is found in several species, mainly in the bronchioles (12). Whether they secrete lipid or protein is unclear. They may have the capacity to produce either lipid or glycoprotein depend ing upon environmental conditions: Airway irritation with smoke or S02 induces transitions of Clara cells to goblet cells and stimulates the produc tion of glycoprotein by these cells (33). A sulfated glycoprotein has been shown to line the luminal surface of airway ciliated cells, including the cilia, in humans and dogs (67), and in cats and geese (30, 56). Its chemical composition is different from that of submucosal gland glycoproteins; it is probably secreted from epithelial cells (22, 24). Evidence for an innervation of epithelial secretory cells is conflicting, and there is probably much species variation. Specific acetylcholinesterase stain ing shows cholinergic efferent fibers in the epithelia of pigs (40): Specific immunofluorescent staining shows adrenergic efferent fibers in the epithelia of pigs (40) and rats (79). Efferent nerve profiles have been observed in the airway epithelia of rats (31) and geese (59): In both these species fibers containing agranular vesicles and fibers containing dense-cored vesicles were found. Some of these fibers in both species were observed near secre tory cells. The presence of a cholinergic innervation of epithelial secretory cells in geese (a species with only epithelial secretory cells and no sub mucosal glands) is confirmed by the finding that tracheal mucus secretion increased following parasympathetic nerve stimulation (59).
Effects of Irritants Acute administration of irritant agents to the airways, including organic vapors (10), ammonia, cigarette smoke, and CS solution (O-chlorobenzili dine malononitrile) (62), increases airway mucus secretion. Inhaled am monia vapor and cigarette smoke stimulate secretion from both epithelial
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secretory cells and submucosal glands, and cause release of a surface muco substance (30, 56). These irritants act partly through reflex neural pathways (perhaps involving stimulation of cough receptors) and partly through a direct effect: The reflex stimulation probably causes secretion from the submucosal glands, and the direct stimulation probably causes secretion from the mucosal cells (56, 62). The mechanism of this direct effect is not clear, but it may involve prostaglandins as intermediaries (62). Chronic administration of S02 (32) or cigarette smoke (34) to the airways increases airway mucus secretion and produces goblet cell hyperplasia, transforma tion of epithelial cells, and enlargement of submucosal glands.
Effects of Drugs Various pharmacologic mediators affect mucus secretion. Histamine, a putative mediator in asthma, stimulated mucus secretion in cats and geese in vivo (62), but had no effect in vitro either in dogs (13) or in humans (68). The discrepancies could be due to species differences. Local application of prostaglandin (PG)El stimulated mucus secretion in various airways of cats (62) and rats (29); also, PGF2a when given locally stimulated mucus secre tion in cats (62), and when inhaled as an aerosol induced sputum production in healthy humans (37). PGF2a stimulates cough receptors, and since part of the effect of this drug on bronchomotor tone is mediated by cholinergic efferent pathways (75), the secretory effects of PGF2a could similarly be mediated partly via cholinergic efferent pathways, although a direct effect cannot be excluded. Tissue inflammation causes local PG release, and such release could account for the increase in mucus secretion found in inflam matory airway diseases. Basic polypeptides (including kallidin and sub stance P) increased tracheal glycoprotein secretion in dogs in vitro, while a kalladin antagonist (hexadrimethrine) decreased it (5). Local application of the anesthetic lidocaine to the tracheas of cats or geese not only blocked the responses to known secretogogues, but also stimulated glycoprotein secretion directly (56, 59). The mechanisms of these actions are unknown but may involve the displacement of calcium ions from extra- and intracel lular binding sites (56). ACTIVE ION TRANSPORT
Since water plays such an important role in the formation of the respiratory tract secretions, we hypothesized that the regulation of water movement into the airway lumen might be important in detennining the physical properties of the mucus layer and the rate of its movement up the airway by the cilia (51). Therefore, we sought methods for the study of water movement across the airway epithelium. It is generally accepted (28) that
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water crosses absorptive (14,1 7,64) and secretory (18, 69) epithelia as a result of local osmotic gradients created by active ion transport. To investi gate ionic fluxes in airway epithelia,we used Ussing's short-circuit current method (71) and pieces of epithelium from the posterior membranous part of dog tracheas. We found a net flux of CI- toward the lumen and a smaller net flux of Na+ toward the submucosa; these net fluxes accounted for the measured short-circuit current (53). Therefore, CI- and Na+ are actively transported across tracheal epithelium and may be the only actively trans ported ions. Our findings have been confirmed subsequently in dogs (2), and suggestive evidence has been provided that CI- and Na+ are transported similarly in rabbits (46) and cats (57) and that Cl- is actively transported toward the lumen in rats (39). Evidence presented for active K+ transport in rabbits (46) is unconvincing. Submucosal gland cells and surface epithelial cells may both be involved in active ion transport. However,since rabbits have no tracheal submucosal glands but their tracheas actively transport CI- and Na+,the sites of active ion movement in this species must be in the surface epithelial cells. Active secretion of CI- may be a property of all epithelial tissues derived from the primitive foregut,since it occurs in the amphibian (27) and mammalian (35) stomach, the mammalian esophagus (60), and the amphibian (25) and mammalian (54) lung. Furosemide reduced net CI- movement toward the lumen when added to the submucosal side,but not when added to the luminal side of the tracheal epithelium (16). Ouabain was bound to more sites on the submucosal mem brane than on the luminal membrane of the tracheal epithelium (77); this binding is presumably to Na+-K+ATPase (4,77),the sodium pump. These findings suggest that active CI- and active Na+ transport occur at the submucosal membrane. CI- and Na+ transport are interdependent,since net CI- movement is greatly reduced by replacement of Na+ in the bathing solution [(2,43); J. H. Widdicombe,personal communication], and by add ing ouabain, an inhibitor of sodium pumps, to the submucosal bathing solution (2,77). In one experimental model for the link between Na+ and CI- movement, CI- enters the cells by a Na+-linked system across the submucosal membranes,the transmembrane Na+ gradient providing the energy for active accumulation of Cl- by the cells. The Na+ entering with the CI- is pumped back to the submucosa by basolaterally placed Na+ pumps,while the CI- diffuses down an electrochemical gradient across the luminal surfaces of the cells. The energy for CI- secretion is thus provided by the transmembrane Na+ gradient and ultimately therefore by the Na+ pump (J. H. Widdicombe,personal communication). The electrical resistances and the spontaneous electrical potential differ ences across epithelia correlate well with physical parameters such as
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permeability to electrolytes, hydraulic conductivity, and the ability of epith elia to maintain an osmotic gradient (21). Airway epithelia of different species have electrical resistances about 300 ohm cm2 and a range of sponta neous electrical potential differences between 20 and 60 mV. This classifies them as "tight junction" epithelia and implies that, relative to "leaky junc tion" epithelia (e.g. rat jejunum), they are less permeable to electrolytes, are poor conductors of water, and can maintain large transepithelial osmotic gradients. Two patterns of electrical potential differences across the luminal and submucosal membranes of rabbit tracheal epithelial cells have been ob served from intracellular recordings. One group found some cells with the inside negative to both the luminal surface and the submucosal surface, and other cells with the inside positive to the luminal surface and negative to the submucosal surface (26). Another group found only cells with the inside negative to both the luminal surface and the submucosal surface, and they attributed the potential differences to the following differences in the per meabilities of each cell membrane to electrolytes: In the luminal membrane the permeability to CI- > S042- > Na+ > K+, and in the submucosal membrane the permeability to K+ > Na+ > CI- > SOi- (46). Further studies of the intracellular distribution of ions and the associated electrical potentials should help to determine the role of active ion transport in transmembrane water movement. We studied the effects of drugs that mimic the actions of the autonomic nervous system, and the effects of mediators on active ion transport across pieces of tracheal epithelium mounted in Ussing chambers. Acetylcholine increased net movement of Cl- and Na+ toward the canine tracheal lumen. The electrical resistance of the tissue was unchanged, and most of the increase of ion movement was electrically neutral. The effect was prevented by small concentrations of atropine (41). Terbutaline, a specific f1z-adrener gic agonist, increased net movement of Cl-, but not of Na+, toward the tracheal lumen in dogs (15) and cats (57). The electrical potential difference increased despite a fall in electrical resistance of the tissue. Propranolol prevented the effects in dogs. The effects of epinephrine (3) were similar to those of terbutaline. Also, terbutaline given intravenously to dogs increased the potential difference between a fluid-filled tracheal segment in situ and the paratracheal fascia (B. Davis, M. G. Marin, J. A. Nadel, unpublished). Phenylephrine, a specific a-adrenergic agonist, increased net movement of CI- and Na+ toward the tracheal lumen in cats (D. Davis, R. J. Phipps, J. A. Nadel, unpublished). The major effect was stimulation of Na+ secretion into the lumen, which was measured under open-circuit conditions. Since phenylephrine had no effect on the electrical properties of the posterior membranous part of tracheal epithelium in dogs (B. Davis, M. G. Marin,
MUCUS SECRETION AND ION TRANSPORT IN AIRWAYS
J.
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A. Nadel, unpublished), the marked effect on the anterior portion of
tracheal epithelium in cats may indicate either a species difference or an effect on submucosal glands, which are abundant in the anterior part of cat tracheas and sparse in the posterior membranous part of dog tracheas. Histamine increased net movement of CI- and Na+ toward the tracheal lumen. The increase in total ion transport was dose-related and could be prevented by an HI-antagonist, but not by an Hz-antagonist (42). The electrical potential difference was increased despite a small decrease in
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tissue resistance. Thus, the effects of autonomic agonists and mediators on the output of respiratory tract secretions could be due in part to their effects on ion transport.
SUMMARY The output of secretions from the airway submucosal glands is regulated by vagal efferent nerves. Stimulation of cough receptors increases mucus output reflexly via the vagus nerves. Adrenergic agonists increase sub mucosal gland secretions in some species, which indicates that adrenergic receptors are present in these cells. However, evidence for adrenergic ner vous pathways to the glands is limited. Irritants and drugs stimulate secre tion from epithelial cells by direct effects. There is also evidence that the secretion of epithelial cells can be stimulated by parasympathetic nervous pathways in birds but not in mammals. Active ion transport of Cl- toward the lumen and of Na+ toward the submucosa results in net ion movement toward the airway lumen in unstimulated tracheal epithelia. Drugs and mediators increase the net movement of ions toward the lumen. No agents have yet been found that increase net ion movement toward the submucosa. The link between ion transport and water secretion in airway epithelia, although speculative, seems likely in view of the evidence from other epith elia. Since airway epithelium is a "tight junction" epithelium, modification of the tight junctions may alter the transepithelial movement of water and ions. We suggest that the depth and consistency of the periciliary layer of airway secretions determine the ability of the cilia to propel the muco protein gel and thereby modify mucociliary transport. To achieve this, secretion of mucus must be controlled separately from the secretion of water. Studies are needed to determine which of the speCialized functions of the epithelial cells interact to regulate the clearance of secretions from the airway. Is the sol maintained by secretion and reabsorption of fluid across the epithelium? Does the sol move with the gel by ciliary action or does it remain stationary? Do changes in the epithelial tight junctions influence net water movement and thus indirectly alter the depth of the sol layer? To answer these questions, techniques are needed to study subunits
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of the airway, including isolated surface cells and submucosal glands; and sensitive methods are required to analyze the very small samples of secre tions for glycoprotein and electrolyte content. Intracellular measurements of electrolyte concentrations and electrical potentials may help to elucidate the mechanisms of transepithelial ion and water movement. The control system for the production and removal of respiratory tract secretions may be altered in disease. For instance, chronic stimulation of cough receptors causes reflex secretion and may be the cause of the hyper plasia of submucosal glands and of the abnormal secretions that occur in chronic bronchitis and asthma (50, 58). The abnormally viscid mucus in cystic fibrosis may be due to a defect in CI- transport, which provides too little water for both the gel and sol layers. These speculations are intended to identify areas for further research, which hopefully will reduce the mor bidity and mortality in these common lung diseases. ACKNOWLEDGMENTS
We thank Ms. Linda Morehead and Mrs. Beth Cost for typing and prepara tion of the manuscript, and Mr. Robert W. Surface for medical illustration. During the preparation of this review, Dr. Davis was supported by Young Investigator Research Award HL-21150 from the National Heart, Lung and Blood Institute. Some of the reported studies were supported by grants from the U.S. Public Health Service: Program Project HL-06285 and Pul monary SCOR Grant HL-19156, and in part by a grant from the Cystic Fibrosis Foundation. Literature Cited 1. Adams, G. K., Aharonson, E. F., Rea sor, M . J., Proctor, D. F. 1976. Collec tion of normal canine tracheobronchial secretions. J. Appl. Physiol. 40:247-49 2. Al-Bazzaz, F., Al-Awqati, Q. 1977. Characteristics of ion transport in ca nine tracheal epithelia. Fed. Proc. 36(3):479 (Abstr.) 3. AI-Bazzaz, F. J., Khan, A., Cheng, E. 1977. Stimulation of chloride secretion across canine tracheal epithelia by theo phylline and epinephrine. Clin. Res. 25:414A (Abstr.) 4. AI-Bazzaz, F. J., Westenfelder, C., Ear nest, W., Kurtzman, N. 1977. Charac terization of canine tracheal epithelium Na-K-ATPase. C/in. Res. 25:413A (Abstr.) 5. Baker, A. P., Hillegass, L. M., Holden, D. A., Smith, W. J. 1977. Effect ofkalli din, substance P, and other basic poly peptides on the production of respira-
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