Mechanisms for Ion and Water Transport across Tracheal Epithelium

Mech. Anal. 1964, 16, 325-353. Gurtin, M. E.; Pipkin, A. C. A General Theory of Heat Conduction with Finite Wave Speeds. Arch. Ration. Mech. Anal. 196...
0 downloads 0 Views 882KB Size
Ind. Eng. Chem. Res. 1992,31, 721-726 Green, A. E.; Rivlin, R. S. Simple Force and Stress Multipoles. Arch. Ration. Mech. Anal. 1964,16,325-353. Gurtin, M. E.; Pipkin, A. C. A General Theory of Heat Conduction with Finite Wave Speeds. Arch. Ration. Mech. Anal. 1968,31, 113. Helsing, J.; Grimvall, G. R. Conductance in Random InductanceCapacitor Networks. Phys. Rev B 1990,31, 113-126. Hinch, E. J. An Averaged-Equation Approch to Particle Interactions in a Fluid Suspension. J.Fluid Mech. 1977,83,695-720. Jeffrey, D. J. On the Averaged-Equation Approach to Conduction through a Suspension. Arch. Mech. 1976,28,423-429. Joseph, D.D.;Preziosi, L. Heat Waves. Rev. Mod. Phys. 1989,61, 41-73; Addendum 1990,62,375-391. Koch, D. L.; Brady, J. F. Nonlocal Dispersion in Porous Media: Nonmechanical Effects. Chem. Eng. Sci. 1987,42, 1377-1392. Landauer, R. Electrical Conductivity in Inhomogeneous Media. In Transport and Optical Properties of Inhomogeneous Media; Garland, J. C., Tanner, D. B., Eds.; AIP Conference Proceedings 40; American Institute of Physics: New York, 1978. Luciani, J. F.; Mora, P. Nonlocal Heat Transport Due to Steep Temperature Gradients. Phys. Rev. Lett. 1983,51,1664-1667. Machta, J. Generalized Diffusion Coefficient in One Dimensional Random Walks with Static Disorder. Phys. Rev. B 1981, 24, 5260-5269. Mindlin, R. D.Microstructure in Linear Elasticity. Arch. Ration. Mech. Anal. 1964,16,51-78. Mindlin, R. D.Second Gradient of Strain and Surface Tension in Linear Elasticity. Znt. J. Solids Struct. 1965,1,417-438.

72 1

Odagaki, T.; Lax, M. Coherent-Medium Approximations in the Stochastic Transport Theory of Random Media. Phys. Rev. B 1981,24,5284-5294. Papanicolaou, G., Ed. Random Media; Springer-Verlag: Berlin, 1987. Phillips, C. G.; Jansons, K. M. The Short-Time Transient of Diffusion outside a Conducting Body. Proc. R. SOC. London, A 1990, 428,431-449. Russel, W. B. Brownian Motion of Small Particles Suspended in Liquids. Annu. Rev. Fluid Mech. 1981,13,425-455. Sahimi, M.; Jerauld, G. R. Random Walks on Percolation Clusters at the Percolation Threshold. J. Phvs. C: Solid State Phvs. 1983. 16,L1043-L1050. Sahimi, M.; Hughes, B. D.; Scriven, L.; Davis, H. T. Stochastic Transport in Disordered Systems. J. Chem. Phvs. 1983.. 73., 6849-6864. Sanchez-Palencia, E., Zaoui, A., Eds. Homogenization Techniques for Composite Media; Springer-Verlag; Berlin, 1987. Silbey, R. Dynamical Processes in Disordered Systems. In Disordered Solids. Structures and Processes; DiBartolo, B.; Oien, G., Collins, J. M., Eds.; Plenum Press: New York, 1989;pp 31-54. Zaoui, A. In Homogenization Techniques for Composite Media, Sanchez-Palencia, E., Zaoui, A., Eds.; Springer-Verlag: Berlin, 1987;part VI. Received for review November 26, 1990 Revised manuscript received March 21, 1991 Accepted April 19, 1991

Mechanisms for Ion and Water Transport across Tracheal Epithelium Irving F. Miller Department of Chemical Engineering (MIC 110), University of Illinois a t Chicago, Chicago, Illinois 60680

T o explain the mechanisms whereby ions and water transport across trachael epithelium, we have developed a new hypothesis in which the principal event is Na+-driven paracellular electroosmotic transport through dynamically controlled tight junctions. Upon stimulation, epithelial cell apical membrane impedance is reduced, resulting in net C1- ion and water flux out of the cells, opening the cation-selective tight junctions, perhaps by activating apical perijunctional actomyosin rings. Na+ flows through the tight junctions from serosa to lumen, in response to an electrochemical gradient developed by transcellular C1- transport from serosa t o lumen, pumping water electroosmotically. The Na+ returns to the serosal side transcellularly, thus completing the circuit. Thus, water transport is directly coupled to Cl- transport, and impaired Cl- transport, as occurs for example in cystic fibrosis, directly results in impaired water transport. Introduction Diseasesof the airways of the lungs are among the major causes of morbidity and mortality in the United States, with approximately 17 million people suffering from chronic bronchitis, asthma, cystic fibrosis, or emphysema. These diseases are often caused or aggravated by such inhaled pollutants as cigarette smoke, sulfur dioxide, or allergens, thus underscoring the role of the lungs as the first line of defense of the body against the effects of inhaled particles, pollutants, and toxicants. Healthy lungs react to environmental challenges primarily by activation of the mucociliary clearance system, shown in the cross section of the epithelial tissue in Figure 1. The respiratory tract ciliated epithelium is covered by a thin layer of fluid consisting of an aqueous periciliary layer bathing the cilia and a blanket, or islands, of viscoelastic mucus (Van As and Webster, 1974). Inhaled pollutants are trapped in the mucus layer, which is propelled by the beating cilia along the periciliary layer up the trachea and out of the lungs. The cilia beat in an asymmetric pattern, with an active stroke in the direction of the mouth, in which the tips of the cilia contact the

mucus layer, and a return stroke in the distal direction, in which the cilia move through the periciliary layer without contacting the mucus. Adjacent cilia beat slightly out of phase, establishing sinusoidal metachronal waves that generally move mouthward up the trachea. The mucociliary clearance system, consisting of the cilia, the mucus, and the periciliary layer, has both a basal and a stimulatory component. When the healthy system is at rest, the cilia beat at a base frequency that, in the dog, ranges over 6-12 Hz (Wong et al., 1986,1988) and exert a force on the mucus layer estimated at 10-l2 N/cilium (Yates et al., 1980). The periciliary layer is maintained at a thickneas about 0.5 pm less than the length of a cilium (about 6 pm), and sufficient mucus is produced to provide a steady movement of the layer up the airways. When the system is stimulated, ciliary beat frequency (CBF),mucin release, and epithelial transport of water and ions all increase to provide an increased mucociliary transport. The physical properties of the mucus and the efficiency of mucociliary clearance from the trachea are controlled by the interaction of several epithelial cell types, some of which perform ion and water transport, while others syn-

0888-5885/92/2631-0721$03.00/00 1992 American Chemical Society

722 Ind. Eng. Chem. Res., Vol. 31, No. 3, 1992

LUMEN

1

RESTING STATE

I

SUBMUCOSA

Figure 1. Cross section of tracheal epithelial tissue. Shown are the epithelial cells, the mucin-producinggoblet cells, the cilia, the mucus layer, and the intercellular junctions, the likely site of the small pores.

Figure 2. Diagram of hypothesis. In the resting state, the small pores are closed and epithelial transport is primarily transcellular. In the stimulated state, the small pores are open, and epithelial transport is primarily paracellular.

thesize and secrete macromolecules (Basbaum, 1984; Basbaum et al., 1981). The overwhelming component of the mucus and the periciliary layer is water, which provides a transport medium and is an active participant in the processes that take place. For example, the rheological properties of the mucus appear to be regulated by its hydration (Shake et al., 1987; Verdugo, 1984), just as its interactions with the cilia are controlled by the volume of periciliary fluid. Thus, the forces exerted on and by the cilia and the maintenance of a healthy mucociliary clearance system and the prevention of lung disease are critically dependent on the control of transport of water into and out of the airways. Tracheobronchial secretions have a normal volume in man estimated at 5-10 mL/day and are about 95% water, 1% protein, 0.9% carbohydrate, and 0.8% lipid (Kaliner et al., 1986; Toremalm, 1962). They are produced predominantly by submucosal glands whose ducts are lined with the ciliated epithelial cells that appear to regulate both the water and the electrolyte concentration of the secretions. In the dog, the tissue formed by the epithelial cells appears to contain two pore populations, respectively, 0.6 and 25 nm in diameter (Man et al., 1986). The far greater numbers of small pores provide approximately 98% of the total area for diffusion. Similar heteropore populations have been reported in alveolar epithelia, though the small wres are larger (Kim and Crandall, 1983). The small pores are probably associated with the tight junctions between the cells, which have been shown to be highly dynamic structures (Gumbiner, 1987). Excised trachael epithelial tissue also appears asymmetric, with osmotic water flow from the apical side less than from the submucosal side, at least when the tissue is in its base state (Man et al., 1984). In addition to providing a physical barrier to transport, the epithelium provides an electrochemical barrier. In the base state, the excised mucosa has been found to have a spontaneous open-circuit potential of approximately 30-40 mV, lumen negative (Al-Bazzaz and Al-Awqati, 1979; Olver et al., 1975; Welsh et al., 1982). This potential yields a short-circuit current of approximately 3 pequiv/ (cm2h) (Al-Bazzazand Al-Awqati, 1979), comprising a measured C1- flux of approximately 2.5 pequiv/(cm2 h) toward the lumen and a measured Na+ flux of approximately 0.5

pequiv/(cm2 h) toward the submucosa (Figure 2). The ion movements appear to be coupled (Levitan, 1979) and have been modeled by an active basolateral Na+-K+ pump removing Na+ from the cell while allowing the passive entry of NaCl (Frizzell et al., 1979; Welsh, 1983a-c; Welsh et al., 1983; Westenfelder et al., 1980; Widdicombe et al., 1979) (co-transport of C1-). This results in the buildup of C1- ion within the cell, leading to passive diffusion of C1- into the lumen across the apical membrane, and the observed open-circuit potential. When the Na+-K+ pump is blocked by M ouabain, net C1- into the lumen is abolished, and the short-circuit current is reduced (Al-Bazzaz and Al-Awqati, 1979; Welsh, 1983a; Widdicombe et al., 1979). On the other hand, isoproterenol has been shown to increase C1- flux and short-circuit current without affecting Na+ flux (Al-Bazzaz and Cheng, 1979; Barthelson et al., 1987; Boucher et al., 1980; Boucher and Gatzy, 1982). Ion fluxes are also subject to manipulation by agents known to affect secretion (Leikauf et al., 1985; Marin et al., 1976, 1977; Nathanson et al., 1983; Smith et al., 1982; Stutts et al., 1988) and CBF (Wong et al., 1986, 1988; Yanaura et al., 1981). Transepithelial ion movements appear to be coupled to water transport (Kim and Crandall, 1982). In human epithelial cell culture, K+ and C1- channels were found important in the regulation of cellular volume change (Hazama and Okada, 1988). In excised dog tracheal epithelium, a mucosal osmotic load led to a decrease in the transepithelial potential difference (Man et al., 1984), a rise in transepithelial conductance (Yankaskas et al., 1987), and the development of a streaming potential (Man and Thomson, 1981), while C1- transport has been linked to fluid transport in canine fetal and neonatal lung (Cotton et al., 1988a,b). Coupling between ions, neutral species, and water flows has also been found in bovine tracheae (Durand et al., 1981, 1984). Water and ions may cross the tracheal epithelium by either a cellular or a paracellular route. Coupled cellular-paracellular ion transport has been reported in which cellular Na+ and C1- fluxes are balanced by paracellular return pathways (Marin and Zaremba, 1979; Stutts et al., 1988; Welsh and Widdicombe, 1980). Stimulation of ion transport and secretion in gallbladder (Cotton and Reuss, 1988; Spring and Ericson, 1982; Tormey, 1977) has been shown to lead to cellular water loss and shrinkage, arguing for a cellular pathway in such secreting epithelia. Similar

Ind. Eng. Chem. Res., Vol. 31, No. 3, 1992 723 cellular effects have been found in canine (Man et al., 1984) and bovine tracheal epithelium (Worman et al., 1986). Paracellular water transport across canine (Widdicombe, 1984) and bovine (Durand et al., 1984) tracheal epithelia has been reported, with some (Widdicombe, 1984; Yankaskas et al., 1987) considering such flows to be driven by a standing osmotic gradient resulting from unstirred layers (Barry, 1981; Diamond and Bossert, 1967). Others suggest direct salt-water coupling via a transcellular solvent drag mechanism (Durand et al., 1984; Hill, 1975a,b; Hill, 1980). An important observation is that tracheal luminal fluid may be hypertonic (Man et al., 1979,1984;Shih et al., 1983; Verdugo, 1984; Yankaskas et al., 1987). At the onset of stimulation, the electrical conductance of the epithelial membrane increases, and the flux of both Na+ and C1- toward the lumen increases (Man et al., 1984, Marin et al., 1976,1977; Welsh et al., 1980; Yankaskas et al., 1987). These changes appear to accompany an increase in the permeability of the apical membranes of the epithelial cells relative to the basolateral membranes (Shorofsky et al., 1983; Welsh et al., 1982, 1983), from a base state in which the apical membrane is less permeable than the basolateral membrane to a stimulated state in which it is more permeable. Presumably, these permeability increases are a result of the opening of specific ion channels in the apical membrane, possibly through the action of a CAMP-dependent protein kinase (Li et al., 1988; McCann and Welsh, 1988). These various observations, the product of many specific and incomparable experiments, have resulted in a fragmented and controversial view of the major mechanisms whereby the tracheal mucociliary clearance system responds to stimuli. What has been lacking is a comprehensive unifying hypothesis that explains all the major observations reported in the literature, fits what is known about the structure and function of epithelial tissue, and yields predictions about how the system responds to stimuli. Herein, such an hypothesis is proposed in which Na+driven electroosmotic transport through dynamically controlled small pores (tight junctions) is the central mechanism controlling stimulated water secretion by the epithelium. This dynamic control is similar to that proposed for rat and hamster intestinal mucosa (Madara and Pappenheimer, 1987; Pappenheimer, 1987; Pappenheimer and Reiss, 1987), where glucose or amino acids in the intestinal lumen activate contraction of perijunctional actomyosin rings in the apical region of epithefial cells, opening tight junctions to permit the transport of nutrients by solvent drag mechanisms, in response to osmotic gradients established by Na+-coupled active transport. It is also similar to one proposed for water secretion by frog skin glands, in which water secretion is directly coupled to Na+ efflux (Neilson, 1990). The hypothesis suggests that stimulation results in the opening of ion channels in the epithelial apical membrane, causing permeability increases in that membrane. These increases allow C1- entry into the lumen, shrink the epithelial cells, and open a paracellular pathway for a Na+-driven electroosmotic flow into the lumen. The Model The model is shown in Figure 2. The tissue is assumed to have two pore populations, the great majority approximately 0.6 nm in diameter, and the rest approximately 25 nm in diameter. Cellular processes control the conductances of the cation-selective small pores, while the large pores are stable and essentially nonselective. In the resting state, the cells forming the pore boundaries are

swollen, and the small pores, consisting of the tight junctions and adjacent cell walls with fixed negative charges formed (probably) from sialic acid residues, are relatively nonconductive. The apical membranes of the cells secrete C1- and absorb Na+ in response to concentration and electrical potential gradients, a result of active Na+ removal via a basolateral Na+-K+ pump accompanied by passive NaCl entry, resulting in accumulation of C1- within the cell. In the resting state, the apical membrane C1- permeability is less than that of the basolateral membrane and the process favors accumulation of C1- within the cell. An electrochemical potential difference across the apical membrane, cell interior electronegative, causes a larger C1secretion from and a smaller Na+ entry into the cells and an electrical potential difference, lumen negative, across the tissue. There may also be a pressure gradient across the apical membrane. In the resting state, transport is primarily transcellular. Small ion and water fluxes may be triggered by forces transmitted by the cilia to the apical surface or by other mechanisms. For example, if water loss causes the mucus to become more viscous or the periciliary layer shallower, the permeability of the apical membrane increases, increasing the net flux of Na+ ions into and C1- ions out of the cell. The resulting net ion and associated osmotic water flux out of the cells and through the large pores compensates for the water loss from the lumen and allows homeostasis to be maintained. In the stimulated state, transport is primarily paracellular. When the tissue is stimulated, CBF increases and the impedance of the apical membrane is reduced to less than that of the basolateral membrane. The resulting net Cl- ion and associated water fluxes shrink the cells and perhaps activate apical perijunctional actomyosin rings, opening the tight junctions. The small pores, lined with fixed negative charges and thus permselective for cations, become more conductive. Na+ flows from serosa to lumen in response to the electrochemical gradient, electroosmotically pumping water. The increased luminal water results in an increased volume of mucus, which is then transported by the stimulated cilia up the trachea. The Na+ returns to the serosal side either transcellularly or through the large pores. Depending on their solute reflection coefficients (which are likely to be small), these relatively nonselective large pores may also provide pathways for osmotic water flow into the lumen and for C1return to the submucosa, thus preventing accumulation of ions on either side of the tissue. Any osmotic flow through the large pores would continue as long as there is an osmotic driving force, controlled by the hyperosmotic electroosmotic flow. When the stimulation causing the water flow ends, the apical membrane resistance increases (perhaps as a result of the forces transmitted by the cilia) and the C1- secretion rate is reduced. The cells reswell, the tight junctions close, and the small paracellular pores become less conductive, returning the tissue to the resting state. Preliminary Experiments

Reliable experimental support for the hypothesis is extremely difficult to obtain. The epithelium is a living tissue that is integrated into a complex system of neural and humoral networks that control ita behavior. If the tissue is removed from the animal for study under controlled conditions, tissue deterioration and separation from ita environment will result in experimental artifacts that will call any experimental results into question. If experiments are conducted in the intact trachea, experimental condi-

724 Ind. Eng. Chem. Res., Vol. 31, No. 3, 1992

tions typically cannot be controlled sufficiently to allow a “clean” experiment to be performed. Over the past several years, we have developed assays that will allow measurements of various aspects of pulmonary clearance to be made in situ in an intact animal. These assays include ways to measure CBF, mucus rheology (viscosity, elasticity, and yield stress), mucus velocity, and the effects of the various neural and humoral pathways that control them. Some of the problems associated with in vitro assays, so pervasive in the literature, are highlighted by our CBF assay (Wong et al., 1986, 1988). For this assay, a laserlight-scattering system was built into a stainless steel probe, which was inserted into the canine trachea. Photons backscattered from the cilia were mixed with laser light from a random depolarizing scatterer. Using the heterodyne mode, autocorrection of the temporal pattern of the detected photons resulted in a measurement of ciliary beat frequency. Using this system, we found that the baseline CBF was in the range 6-12 Hz, well below the values reported in the literature for invasively measured CBF (Yanaura et al., 1981). During excision of the trachea for in vitro experiments, we found that CBF increased to about 14-20 Hz, consistent with measurements reported in the literature. Apparently, the act of excising tracheal tissue stimulates CBF, and a true resting state cannot be measured in an in vitro experiment. Electroosmosis. For electroosmosis,measurements are typically made of volume flows in response to electric potential gradients. In 1971, we (Breslau and Miller, 1971) measured electroosmoticflows across a series of cation- and anion-exchange membranes, from which we developed a hydrodynamic model for electroosmosis that, for the first time, quantitatively fit experimental data and provided detailed correlations between membrane structure and transport properties. It suggested that such ion-exchange membranes behave like a porous structure with the pores much more like parallel flat plates than like circular cylinders, which was the model widely accepted at the time. In addition, ions within the pores appear to be less hydrated than they are in free solution, and pore water appears to be more highly structured than is free water. It also showed that electroosmotic flows are extremely strong functions of the ratio of pore diameter to driving ion diameter, with electroosmotic coefficients asymptotically approaching infinity as the ratio approaches unity. Unfortunately, the experimental procedure used in these studies, involving the separation of two fixed-volume stirred chambers by the membrane, cannot be used with an intact tracheal epithelium. However, the studies allowed some estimates of the behavior of epithelial tissue to be made. The ion-exchange membrane data and the hydrodynamic model were used to estimate relative pore conductance, electroosmotic coefficients, and reflection coefficients for Na+ in 0.6-and 25-nm pores, the estimated size of the pores in canine tracheal epithelium. The calculated Na+ reflection coefficients were 0.89 (0.6 nm) and 0.02 (25 nm), verifying the permselectivity of the small pores and the lack of selectivity of the large pores. The estimated electroosmotic coefficient for Na+ in a 0.6-nm pore was 12.5 mol of water/Faraday. An increase in small-pore diameter of 5% resulted in a decrease in Na+ reflection coefficient of 8%, a decrease in Na+ electroosmotic coefficient of 12%, but an increase in pore conductance of 51%. Thus, very small changes in small pore diameter appear to have a very large effect on water flow, allowing very sensitive control of secretion rates, in keeping with the hypothesis.

Although this preliminary result supports the hypothesis, what is required is a direct experimental verification using intact tracheal epithelium. The need for such experimental verification is underscored by the measurements of Na+-driven water secretion in frog skin glands, reported to be in the range of 215 mol of water/Faraday (Neilson, 1990). Considering the asymptotic rise of electroosmotic coefficient as the ratio of pore size to transporting ion size approaches unity, this large value for frog skin, as compared to the estimate used in the model, could easily be accounted for by small differences in pore size among epithelial tissues from different species. Mucus Properties. Samples of canine tracheal mucus was collected from a tracheal pouch (Wardell et al., 1970), produced when a 5-6-cm segment of the cervical trachea was separated and formed in situ into a subcutaneous buried closed organ independent of the remaining trachea, whose cut ends were reconnected. The pouch has been shown to be an independent, histologically normal, physiologically functional, autonomically innervated portion of the corresponding intact trachea and is suitable for long-term studies. Eighteen mucus samples, collected from the pouch approximately weekly, were analyzed for Na+ (flame photometer), C1- (Chloridimeter), and osmolarity (dew-point osmometer). Concentrations of Na+ were significantly lower than those of Cl-, and there was an inverse correlation between C1- content and osmolarity, in keeping with the hypothesis that predicts that transcellular water flux is directly driven by C1-, while paracellular water flux is indirectly driven by C1- through its control of small-pore conductance. In a series of preliminary experiments, Na+ (flame photometer), C1- (Chloridimeter), and osmolarity (dewpoint osmometer) were measured for several samples of mucus obtained during routine examinations of patients with cystic fibrosis. Cystic fibrosis is characterized by mucus that is more viscous and adhesive than normal and, as a result, is more difficult to remove from the airways than is normal mucus. It has recently been demonstrated that the disease is characterized by a gene defect that affects the C1- transport system in the cell membrane (Rommens et al., 1989; Willumsen and Boucher, 1989). Our measurements indicated that the cystic fibrosis mucus showed reduced C1- content and increased osmolarity, in keeping with the hypothesis that water transport is primarily controlled by C1- transport. Using partially dehydrated canine pouch mucus as a model, we also have preliminary results indicating that the increased viscosity and adhesiveness of cystic fibrosis mucus is a direct result of its reduced water content, brought about by the C1transport defect (Pillai et al., 1990). Discussion Mucociliary transport is a very complex dynamic process and is under very tight control. For example, if the periciliary layer extends beyond the ends of the cilia, the cilia will not contact the mucus layer, and the mucus will not move up the trachea. If the periciliary layer is so shallow that the ends of the cilia extend into the mucus layer by as much as 1 pm, the cilia will stick in the mucus layer, and the mucus layer will not move up the trachea. The diameter and surface area of the airways varies over several orders of magnitude through the pulmonary tree, and the secretion rates vary both in position and in time. In spite of these variations, the healthy lungs maintain open airways with a mobile mucus layer that clears the lungs of inhaled materials and keeps the airways free for gas-inhaled materials and keeps the airways free for gas transport, under highly varied and unpredictable conditions.

Ind. Eng.Chem. Res., Vol. 31, No. 3, 1992 725 The control of such a complex and dynamic system necessarily must be very sophisticated and sensitive to small perturbations. Electroosmosis is a mechanism that can provide the necessary dynamic regulation for the secretory system. Since, in electroosmosis, pore conductance is such a sensitive nonlinear function of pore diameter, a very small change in pore diameter would produce a very large change in conductance. Thus, control of electroosmotic water transport by control of pore diameter is a highly sensitive system. The hypothesis suggests that pore diameter itself is controlled by the conductance of the apical membrane of the epithelial cells. The cilia that drive the mucus are themselves attached to the apical membrane. Thus, the system that controls the secretion of water and the system that controls mucus transport are located in the same place. This dual location at the apical membrane implies that a control system for mucociliary transport may be located a t the apical membrane. Such as electroosmotic mechanism may also be at work in the rat and hamster intestinal mucosa studied by Pappenheimer et al. (Madara and Pappenheimer, 1987; Pappenheimer, 1987; Pappenheimer and Reiss, 1987). Although these authors consider the Na+-coupledtransport mechanism to be osmotic in nature, we have shown that glucose, at least, can be transported across membranes by Na+-coupled electroosmosis (Ryu and Miller, 1971). To accommodate such solutes as glucose and amino acida that are transported across the intestinal mucosa, the pores would have to be larger than those in the pulmonary epithelium. Pappenheimer et al. estimate that the intestinal tight junctions are about 5 nm in diameter, approximately 10 times the diameter of the small pores in the pulmonary epithelium. Although the hypothesized mechanism is plausible, it has not yet been proven. Such proof requires carefully designed experiments on intact animal models, experiments that are currently beyond the capability of workers in this field. It is possible that experiments using the canine pouch model may provide the necessary testa of the hypothesis and lead to further insights into how the mucociliary transport system functions. Acknowledgment

Measurements of mucus composition and properties were performed by R. Pillai and T. Chandra. L. B. Wong and D. B. Yeates have been my collaborators in all the mucociliary clearance work cited. They have also provided many helpful insights during the development of the hypothesis. Registry No. Cl-,16887-00-6; Na+, 7440-23-5; water, 7732-185.

Literature Cited Al-Bazzaz, F. J.; Al-Awqati, Q. Interaction Between Sodium and Chloride Transport in Canine Tracheal Mucosa. J. Appl. Physiol. 1979,46, 111-119. Al-Bazzaz, F. J.; Cheng, E. Effect of Catecholamines on Ion Transport in Dog Tracheal Epithelium. J. Appl. Physiol. 1979, 47, 397-403. Barry, P. H. Unstirred Layers and Volume Flows Across Biological Membranes In: Ussing, H. H., Bindsley, N., Lassen, N. A., Sten-Knudsen, O., Eds. Water Transport Across Epithelia, Alfred Benzon Symposium 151 Munksgaard Copenhagen, 1981; pp 132-153. Barthelson, R. A,; Jacoby, D. B.; Widdicombe, J. H. Regulation of Chloride Secretion in Dog Tracheal Epithelium by Protein Kinase C. Am. J. Physiol. 1987,253, C802-C808.

Basbaum, C. B. Regulation of Secretion from Serous and Mucous Cells in the Trachea. Ciba Foundation Symp. 109, Mucus and Mucosa; Pitman: London, 1984, pp 4-19. Basbaum, C. B.; Ueki, I.; Brezina, L.; Nadel, J. A. Tracheal Submucosal Gland Serous Cella Stimulated In Vitro with Adrenergic and Cholinergic Agents. Cell Tissue Res. 1981, 220, 481-498. Boucher, R. C.; Gatzy, J. T. Regional Effects of Autonomic Agents on Ion Transport Across Excised Canine Airways. J.App. Physiol. 1982,52, 893-901. Boucher, R. C., Jr.; Bromberg, P. A.; Gatzy, J. T. Airway Transepithelial Electric Potential In Vitro: Species and Regional Differences. J. Appl. Physiol. 1980,48, 169-176. Breslau, B. R.; Miller, I. F. A Hydrodynamic Model for Electroosmosis. Ind. Eng. Chem. Fundam. 1971,10, 554-564. Cotton, C. U.; Reuss, L. Effect of Changes in Mucosal C1- or K+ Concentration on Cell Volume in Necturus Gallbladder Epithelium. FASEE J. 1988,2, A1726. Cotton, C. U.; Boucher, R. C.; Gatzy, J. T. Bioelectric Properties and Ion Transport Acroas Excised Canine Fetal and Neonatal Airways. J. Appl. Physiol. 1988a, 65, 2367-2375. Cotton, C. U.; Boucher, R. C.; Gatzy, J. T. Paths of Ion Transport Across Canine Fetal Tracheal Epithelium. J. Appl. Physiol. 1988b, 65, 2376-2382. Diamond, J. M.; Bossert, W. H. Standing-Gradient Osmotic Flow: A Mechanism for Coupling of Water and Solute Transport in Epithelia. J. Gen. Physiol. 1967, 50, 2061-2083. Durand, J.; Durand-Arczynska, W.; Haab, P. Volume Flow, Hydraulic Conductivity and Electrical Properties Across Bovine Tracheal Epithelium In Vitro: Effect of Histamine. Pflugers Arch. 1981,392, 40-45. Durand, J.; Durand-Arczynska, W.; Vulliemin, P. Current-Induced Volume Flow Across Bovine Tracheal Epithelium: Evidence for Sodium-Water Coupling. J. Physiol. 1984, 348, 19-34. Frizzell, R. A.; Field, M.; Schultz, S. G. Sodium-Coupled Chloride Transport by Epithelial Tissues. Am. J. Physiol. 1979,236, F1F8. Gumbiner, B. Structure, Biochemistry, and Assembly of Epithelial Tight Junctions. Am. J. Physiol. 1987, 253 (Cell Physiol. 22) C7494758. Hazama, A.; Okada, Y.Caz+Sensitivity of Volume-Regulatory K+ and C1- Channels in Cultured Human Epithelial Cells. J.Physiol. 1988,402,687-702. Hill, A. E. Solute-Solvent Coupling in Epithelia: A Critical Examination of the Standing-Gradient Osmotic Flow Theory. Proc. R. SOC. London, E. 1975a, 190, 99-114. Hill, A. E. Solute-Solvent Coupling In Epithelia: An Electro-Osmotic Theory of Fluid Transfer. Proc. R. SOC. London, E 1975b, 190, 115-134. Hill, A. E. Salt-Water Coupling in Leaky Epithelia. J. Membr. Eiol. 1980,56, 177-182. Kaliner, M.; Shelhamer, J. H.; Borson, B.; Nadel, J.; Patow, C.; Maron, Z. Human Respiratory Mucus. Am. Reo. Respir. Dis. 1986,134,612-621. Kim, K.-J.; Crandall, E. D. Effects of Exposure to Acid on Alveolar Epithelial Water and Solute Transport. J. Appl. Physiol. 1982, 52,902-909. Kim, K.-J.; Crandall, E. D. Heteropore Populations of Bullfrog Alveolar Epithelium. J. Appl. Physiol. 1983, 54, 140-146. Leikauf, G. D.; Ueki, I. F.; Nadel, J. A.; Widdicombe, J. H. Bradykinin Stimulates C1 Secretion and Prostaglandin Ez Release by Canine Tracheal Epithelium. Am. J.Physiol. 1985,248,F48-F55. Levitan, I. B. The Basic Defect in Cystic Fibrosis. Science 1989,244, 1423. Li, M.; McCann, J. D.; Leidtke, C. M.; Nairn, A. C.; Greengard, P.; Welsh, M. J. CAMP-Dependent Protein Kinase Opens Chloride Channels in Normal But Not Cystic Fibrosis Airway Epithelium. FASEB J. 1988,2, A1724. Madara, J. L.; Pappenheimer, J. R. Structural Basis for Physiological Regulation of Paracellular Pathways in Intestinal Epithelia. J. Membr. Eiol. 1987, 100, 149-164. Man, S. F. P.; Thomson, A. B. R. Nonelectrolyte Permeability of Canine Tracheal Epithelium. J. Appl. Physiol. 1981,51,363-368. Man, S . F. P.; Adams, G. K., 111; Proctor, D. F. Effects of Temperature, Relative Humidity, and Mode of Breathing on Canine Airway Secretions. J. Appl. Physiol. 1979, 46, 205-210. Man, S. F. P.; Hulbert, W.; Park, D. S. K.; Thomson, A. B. R.; Hogg, J. C. Asymmetry of Canine Tracheal Epithelium: Osmotically Induced Changes. J. Appl. Physiol. 1984,57, 1338-1346.

726 Ind. Eng. Chem.Res.,Vol. 31, No. 3, 1992 Man, S. F. P.; Hulbert, W. C.; Mok, K.; Ryan, T.; Thomson, A. B. R. Effects of Sulfur Dioxide on Pore Populations of Canine Tracheal Epithelium. J . Appl. Physiol. 1986, 60, 416-426. Marin, M. G.; Zaremba, M. M. Interdependence of Na+ and C1Transport in Dog Tracheal Epithelium. J . Appl. Physiol. 1979, 47, 598-603, Marin, M. G.; Davis, B.; Nadel, J. A. Effect of Acetylcholine on Cland Na+ Fluxes Across Dog Tracheal Epithelium In Vitro. Am. J . Physiol. 1976, 231, 1546-1549. Marin, M. G.; Davis, B.; Nadel, J. A. Effect of Histamine on Electrical and Ion Transport Properties of Tracheal Epithelium. J . Appl. Physiol. 1977,42, 735-738. McCann, J. D.; Li, M.; Welsh, M. J. Identification and Regulation of Whole-CellChloride Currents in Human Tracheal Euithelium. FASEB J . 1988,2, A1723. Nathanson. I.: Widdicombe. J. H.: Barnes. P. J. Effect of Vasoactive Intestinal Peptide on Ion Trhsport Across Dog Tracheal Epithelium. J . Appl. Physiol. 1983, 55, 1844-1848. Neilson, R. Isotonic Secretion Via Frog Skin Glands In Vitro. Water Secretion Is Coupled to the Secretion of Sodium Ions. Acta Physiol. Scand. 1990,139, 211-221. Olver, R. E.; Davis, B.; Marin, M. G.; Nadel, J. A. Active Transport of Nat and Cl- Across the Canine Tracheal Epithelium In Vitro. Am. Rev. Respir. Dis. 1976,112, 811-815. Pappenheimer, J. R. Physiological Regulation of Transepithelial Impedance in the Intestinal Mucosa of Rats and Hamsters. J . Membr. Biol. 1987, 100, 137-148. Pappenheimer, J. R.; Reiss, K. Z. Contribution of Solvent Drag Through Intercellular Junctions to Absorption of Nutrients by the Small Intestine of the Rat. J . Membr. Biol. 1987,100,123-136. Pillai, R.; Chandra, T.; Miller, I. F.; Yeatea, D. B. C1- Concentration, Hydration, and the Rheological Properties of Airway Mucus. FASEB J . 1990,4(4), A1092. Rommens, J. M.; Iannuzzi, M. C.; Kerem, B.; Drumm, M. L.; Melmer, G.; Dean, M.; bzmahel, R.; Cole, J. L.; Kennedy, D.; Hidaka, N.; Zsiga, M.; Buchwald, M.; Riordan, J. R.; Tsui, L.-C.; Collins, F. S. Identification of the Cystic Fibrosis Gene: Chromosome Walking and Jumping. Science 1989,245, 1059-1080. Ryu, S. M.; Miller, I. F. Solute Transport Across Polyelectrolyte Complex Membranes. J . Biomed. Mater. Res. 1971,5,287-306. Shake, M. P.; Dresdner, R.; Gruenauer, L.; Yeates, D. B.; Miller, I. F. A Direct Measuring Capillary Viscoelastimeter for Mucus. Biorheology 1987,24,231-235. Shih, C. KO; Chakrin, L. W.; Litt, M. A Kinetic Model of Aqueous Phase Resorption from the Trachea. AZChE Symp. Ser. 227 1983, 79, 139. Shorofsky, S. R.; Field, M.; Fozzard, H. A. Electrophysiology of C1 Secretion in Canine Trachea. J . Membr. Biol. 1983,72, 105-115. Smith, P. L.; Welsh, M. J.; Stoff, J. S.; Frizzell, R. A. Chloride Secretion by Canine Tracheal Epithelium: I. Role of Intracellular CAMPLevels. J . Membr. Biol. 1982, 70, 217-226. Spring, K. R.; Ericson, A.-C. Epithelial Cell Volume Modulation and Regulation. J . Membr. Biol. 1982, 69, 167-176. Stutts, M. J.; Gatzy, J. T.; Boucher, R. C. Effects of Metabolic Inhibition on Ion Transport by Dog Bronchial Epithelium. J. Appl. Physiol. 1988,64, 253-258. Toremalm, N. G. The Daily Amount of Tracheo-Bronchial Secretions in Man. Acta Oto-Laryng. Suppl. 1962, 158, 43-53. Tormey, J. McD. Anatomical Methods for Studying Transport Across Epithelia. In: Jungreis, A. M., Hodges, T. K., Kleinzeller, A., Schultz, S. G., Eds. Water Relations in Membrane Transport in Plants and Animals; Academic Press: New York, 1977; pp 233-248. Van As, A,; Webster, I. The Morphology of Mucus in Mammalian Pulmonary Airways. Environ. Res. 1974, 7, 1-12.

Verdugo, P. Hydration Kinetics of Exocytosed Mucins in Cultured Secretory Cells of the Rabbit Trachea: A New Model. Ciba Foundation Symp. 109, Mucus and Mucosa; Pitman: London, 1984; pp 212-234. Wardell, J. R., Jr.; Chakrin, L. W.; Payne, B. S. The Canine Tracheal Pouch. A Model for Use in Respiratory Mucus Research. Am. Rev. Respir. Dis. 1970, 101, 741-754. Welsh, M. J. Evidence for Basolateral Membrane Potassium Conductance in Canine Tracheal Epithelium. Am. J . Physiol. 1983a, 244, C3774384. Welsh, M. J. Intracellular Chloride Activities in Canine Tracheal Epithelium. Direct Evidence for Sodium-Coupled Intracellular Chloride Accumulation in a Chloride-Secreting Epithelium. J . Clin. Invest. 198313, 71, 1392-1401. Welsh, M. J. Inhibition of Chloride Secretion by Furosemide in Canine Tracheal Epithelium. J . Membr. Biol. 19&, 71,215F226. Welsh, M. J.; Widdicombe, J. H. Pathways of Ion Movement in the Canine Tracheal Epithelium. Am. J . Physiol. 1980,239, F215F221. Welsh, M. J.; Widdicombe,J. H.; Nadel, J. A. Fluid Transport Across the Canine Tracheal Epithelium. J . Appl. Physiol. 1980, 49, 905-909. Welsh, M. J.; Smith, P. L.; Frizzell, R. A. Chloride Secretion by Canine Tracheal Epithelium: 11. The Cellular Electrical Potential Profile. J. Membr. Biol. 1982, 70, 227-238. Welsh, M. J.; Smith, P. L.; Frizzell, R. A. Chloride Secretion by Canine Tracheal Epithelium: 111. Membrane Resistances and Electromotive Forces. J . Membr. Biol. 1983, 71, 209-218. Westenfelder, C.; Earnest, W. R.; Al-Bazzaz, F. J. Characterization of Na-K-ATPase in Dog Tracheal Epithelium: Enzymatic and Ion Transport Measurements. J . Appl. Physiol. 1980, 48, 1008-1019. Widdicombe, J. H. Fluid Transport Across Airway Epithelia. Ciba Foundation Symp. 109, Mucus and Mucosa; Pitman: London, 1984; pp 109-120. Widdicombe, J. H.; Ueki, I. F.; Bruderman, I.; Nadel, J. A. The Effects of Sodium Substitution and Ouabain on Ion Transport by Dog Tracheal Epithelium. Am. Rev. Respir. Dis. 1979, 120, 385-392. Willumsen, N. J.; Boucher, R. C. Activation of an Apical C1- Conductance by Ca2+Ionophores in Cystic Fibrosis Airway Epithelia. Am. J . Physiol. 1989,256 (Cell Physiol. 25) C2264233. Wong, L. B.; Miller, I. F.; Yeates, D. B. Measurement of Ciliary Beat Frequency in a Two-sided Chamber. Proc. 39th ACEMB 1986, 28, 254. Wong, L. B.; Miller, I. F.; Yeates, D. B. Stimulation of Ciliary Beat Frequency by Autonomic Agonists: In Vivo. J . Appl. Physiol. 1988,65, 971-981. Worman, H. J.; Brasitus, T. A.; Dudeja, P. K.; Fozzard, H. A.; Field, M. Relationship between Lipid Fluidity and Water Permeability of Bovine Tracheal Epithelial Cell Apical Membranes. Biochemistry 1986,25, 1549-1555. Yanaura, S.; Imamura, N.; Misawa, M. Effects of Expectorants on the Canine Tracheal Ciliated Cells. Jpn. J. Pharrnacol. 1981,31, 951-965. Yankaskas, J. R.; Gatzy, J. T.; Boucher, R. C. Effects of Raised Osmolarity on Canine Tracheal Epithelial Ion Transport Function. J . Appl. Physiol. 1987, 62, 2241-2245. Yates, G. T.; Wu, T. Y.; Johnson, R. E.; Cheung, A. T. W.; Frand, C. L. A Theoretical and Experimental Study on Tracheal MucoCiliary Transport. Biorheology 1980, 17, 151-162. Received for review December 3, 1990 Revised manuscript received March 15, 1991 Accepted March 29, 1991