Sodium Ion Transport and Airway Mucus Secretion

secreting cell. Use of Ussing-like chambers offers the advantage that the electrical properties of tissue can be monitored throughout the culture period as an index of tissue viability/ but this technique limits the amount of tissue that can be placed in culture. Ideally, each explant system should be thoroughly characterized with respect to secretory function. For example, explanted tissue should show a physiologic dose-dependence of the secretory response to known secretagogues. In addition, strong supporting evidence for intactness of explanted tissue can be derived from the demonstration of biochemical events related to the stimulus for secretion of mucous glycoproteins. For example, as demonstrated by Dr. Liedtke in this conference, ,B-adrenergic agonists not only trigger the release of mucin from cat tracheal explants, but they increase tissue levels of cAMP and promote binding of endogenous cAMP to specific proteins. Results such as these strongly support the conclusion that the release of mucous glycoprotein is a physiologic response to ,8adrenergic agonists. CoNCLUSION

Other issues concerning the assessment of mucous glycoprotein secretion by explants of airways epithelium also assume considerable importance. Most techniques do not permit determination of the cell-of-origin of secreted glycoproteins. Use of autoradiographic techniques, while potentially helpful in this area, also suffer from non-specificity of labelling and do not afford an opportunity to characterize the labelled secretory product chemically. Advances in cell isolation technology should provide an opportunity to directly relate secretory products to their cell-of-origin. In summary, two approaches are required to advance the state of the art in the investigative area of respiratory tract mucous glycoprotein secretion. The first is to develop and use analytical techniques which are reliable and specific for the secretory macromolecule. The second is to develop new tissue and cell preparations with which we can begin to answer questions about the cells-of-origin of secreted glycoproteins, and about differences, if any, between the mechanisms of secretion of each secretory cell type. REFERENCES

1 Boat TF, Cheng PW. Biochemistry of airway mucus secretions. Fed Proc 1980; 39:3067-74 2 Roussel P, Degand P, Lamblin G, Laine A, LaFitte JJ. Biochemical definition of human tracheobronchial mucus. Lung 1978; 154:241-60 4 Cheng PW, Sherman JM, Boat TF, Bruce M. Analytical biochemistry (in press) 5 Neutra MR, Grand RJ, Trier JS. Glycoprotein synthesis, transport, and secretion by epithelial cells of human rectal mucosa. Lab Invest 1977; 36:535-46 6 Boat TF, Kleinerman Jl, Fanaroff AA, Matthews LW. Toxic effect of oxygen on cultured neonatal respiratozy epithelium. Pediat Res 1973; 7:607 7 Phipps RJ, Nadel JA, Davis B. Effect of alpha-adrenergic stimulation on mucus secretion and on ion transport in cat trachea in vitro. Am Rev Respir Dis 1980; 121:359-65

CHEST, 81: 5, MAY, 1982 SUPPLEMENT

Sodium lon Transport and Airway Mucus Secretion"' The Effect of the Sodium lonophore Monensin on Glycoprotein Secretion by Cultured Rat Trachea ]. R. Etchison, Ph.D.; T. Kaizu, Ph.D .; R. C. Frates, ]r., M.D.; ]. A. Last, Ph.D.; and C. E. Cross, M.D., F.C.C.P.

Ithough the mucous glycoprotein component is thought to be the major factor in determining the physical characteristics of respiratory tract mucus, details concerning the cellular mechanisms and regulatory control of mucus secretion are incomplete. Much of our knowledge of the constituent macromolecules of bronchial secretions derives from studies of sputum from patients with various diseases characterized by mucus hypersecretion. 1-4 While these studies have yielded information on the biochemical structure of some of the glycoprotein constituents, there have been few studies which have focused on the mechanisms of intracellular mucus glycoprotein biosynthesis. Investigation of the mechanisms of respiratory tract mucus glycoprotein assembly, processing, and secretion may be expected to lead to new insights into and a better understanding of the cellular basis of such diseases as cystic fibrosis, bronchial asthma, and chronic bronchitis. The standardization and refinement of techniques for the in vitro organ culture of tracheal explants over the last few years have made possible studies to characterize and quantify the effects of a large number of substances which modulate airway mucus glycoprotein release."·7 In addition, we have utilized this in vitro model system to quantify the effects of in vivo exposure to various noxious inhalants."·" The tracheal explant organ culture system also provides a biochemically manipulable system to investigate and characterize the mechanisms of mucus glycoprotein biosynthesis and secretion. In this report we describe studies to investigate the effects of the sodium ionophore monensin on the assembly and release of mucous glycoproteins by rat tracheal explants. Monensin, which binds sodium ions more avidly than potassium ions, has been shown to perturb glycoprotein secretion by several mammalian cell lines (including plasma cells), apparently by interfering with secretory processes at the level of the Golgi complex, possibly as a result of the preferential partitioning of the ionophore into low density, lipid-enriched internal membranes. 10"12 Our hypothesis in these studies was that perturbations in the Na + /K + ion gradients in the Golgi complex would dissipate the driving force for glycoprotein assembly and processing and that different glycoprotein constituents and/ or different cell types engaged in the synthesis of the glycoproteins might be differentially affected. Our results show that monensin

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Department of Internal Medicine and California Primate Research Center, University of California, Davis. Reprint requests: Dr. Etchison, California Primate Research

Center, University of California, Davis 95616

LUNG FLUIDS AND SECRETIONS 31S

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labelled precursors into glycoproteins secreted by rat tracheal explants. ( [l-•-fl) (3H]leucine; ( 0-0) [3 H]glucosamine; and, ( •-•) pss]sulfate. dramatically inhibited the secretion of glycoproteins by rat tracheal explants and that high molecular weight, sulfated glycoproteins were preferentially affected at low concentrations of monensin. MATERIAL AND METHODS

The conditions for the preparation and culture of rat trachea were exactly as described previously.s Explant sections (70 to 75 mm 2 ) were prepared from the lower half of the trachea from freshly killed two-month old COPD-free Sprague-Dawley rats. Incubations in media containing different concentrations of monensin and containing either 25 ILCi/ml (3H]glucosamine, 50 ILCi/ml (35S]sulfate, or 1 ILCi/ml (3H]Ieucine were carried out in triplicate. All incubations were for 22 hr at 37°C in an atmosphere of 5% C0 2 /95% air saturated with water. Both the radioisotope precursors and the monensin were present for the duration of the incubation. Quantitation of radiolabelled glycoproteins secreted was performed by assaying portions of each sample for TCA-precipitable radioactivity as described previously.9 The remainder of the samples was dialyzed exhaustively against distilled water at 4 'C and lyophilized. The lyophilized samples were redissolved, reduced with dithiothreitol, and alkylated with iodoacetamide as described by Boat et aJ.ts The reduced and alkylated samples were then analyzed by gel filtration through a 1.5 X 50 em column of sepharose CL-4B equilibrated and eluted with 6 M urea in 0.5 M TrisHC1, pH 8.1, containing 0.01 M dithiothreitol. The column was eluted at a How rate of .20 ml/hr and 1 ml fractions were collected. Radioactivity was assayed directly by counting a portion of each fraction in an emulsifying scintillation cocktail.

32S 24TH ASPEN LUNG CONFERENCE

The effects of different concentrations of monensin on the secretion of radiolabelled glycoproteins into the culture medium is shown in Figure 1. A 5oo; reduction in both glucosamine and sulfate incorporation into the TeA-precipitable secreted glycoproteins is evident at a concentration of 0.1 nmol/ml monensin and maximal inhibition ( aoo;) occurred between 1 and 2 nmol/ml. By contrast, incorporation of radiolabelled leucine into TCA-precipitable material was only slightly affected at these concentrations. As a check of the reliability of the TCA-precipitation methods used in the above and previous studies, the samples were also assayed for radioactivity remaining after exhaustive dialysis against distilled water. Similar results were obtained by both methods and the results indicated that at least 80% of the non-dialysable radioactivity was TCA-precipitable. Furthermore, assay of the dialysis bags after these dialyses indicated that there was no significant problem with adsorption of radiolabelled material onto the dialysis bags. To further characterize the effects of monensin on the secretion of glucosamine and sulfate-labelled glycoproteins, the samples from the experiment described above were reduced and alkylated 13 and chromatographed on sepharose CL-4B. Figure 2 shows the gel filtration profiles obtained from control tracheal explants and tracheal explants incubated in the presence of different concentrations of monensin. Panels A, B, and C are glucosamine-labelled glycoproteins, while panels D, E, and F are sulfate labelled glycoproteins. A major portion of the glucosamine-labelled glycoproteins and virtually all of the sulfate labelled glycoproteins elute in or near the column void volume. In addition, a large portion of the glucosamine labelled glycoproteins elute as a heterogeneous population with apparent molecular weights ranging from about 50,000 daltons to 200,000 or 300,000 daltons. It is evident from the data in Figure 2 that the large molecular weight (greater than 500,000 daltons) sulfated glycoproteins eluting in or near the column void volume are more sensitive to the inhibitory effects of monensin than are the non-sulfated glycoproteins. Since the monensin and the radiolabelled precursors were both present for the duration of the incubations ( 22 hr), these results reflect inhibition presumably by a secretory blockade at the level of processing in the Golgi complex rather than an inhibition of release from storage granules. Further studies are needed to determine the kinetics of this inhibition by monensin and to determine whether this inhibition occurs selectively in certain cell types. The mechanism whereby a specific class of glycoproteins are subject to this secretory blockade while others are relatively unaffected is not known and deserves further study. These results support the concept that the transport and secretion of tracheal mucus glycoproteins are significantly affected by monensin, an ionophore that alters intracellular transport of Na + ions. The interpretation of these results with respect to actual airway mucus biosynthesis, transport, and secretion is not clear. Studies

CHEST, 81: 5, MAY, 1982 SUPPlEMENT

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of secretory glycoproteins in a variety of cellular systems indicates that the transfer takes place in a sequential fashion from cytoplasm, into the cisternae of the rough endoplasmic reticulum, to the Golgi complex, to the plasma membranes, and thence to the cellular exterior. Different steps of this processing could be influenced by monensin and the results presented here do not indicate at which stage of this processing the observed inhibition by monensin is occurring. Inhibition at any of these stages of synthesis, processing, and secretion, alone or in combination, could result in decreased mucus secretion. The most likely explanation, in terms of cellular mechanisms, relates to the influences of monensin on Na + transport across intracellular membranes and, in particular, the perturbation of the secretory process at the level of the Golgi complex as has been reported for a variety of cell types in tissue culture. 10-12 In studies of secretion by pancreatic acinar cells, Tartakoff and VassallP 2 have shown that transit from the Golgi appears to be the only stage of the secretory process that is affected by monensin; regulated secretion from mature secretory granules was not affected. As a result of the secretory blockade, one might surmise there would be an increase in intracellular glyco-

CHEST, 81: 5, MAY, 1982 SUPPLEMENT

proteins corresponding to the secretory decrease. In the above studies, the tracheal tissues were extracted after the incubation and the cell-associated glycoproteins were also quantified. The results were similar to the inhibition of secreted glycoproteins. Several explanations for this failure to observe an increase in cell-associated glycoproteins are possible. During the long incubation times used in these initial studies, inhibition of synthesis may occur as a result of the blockade. Alternatively, and more likely, increased degradation of the accumulating glycoproteins may occur as a result of the aberrant accumulation of these glycoproteins. Further studies, including shorter incubation times with increased concentrations of the radiolabel precursors, should help resolve this question. One thrust of recent studies of the mechanisms regulating mucus glycoprotein secretion by tracheal (and bronchial) explants is to understand the underlying pathophysiology of diseases that express themselves at a multiorgan level, such as cystic fibrosis. Our understanding of these processes in cell biological and molecular terms will increase as we continue to unravel the details of the normal processes regulating glycoprotein secretion by cells in vivo.

LUNG FLUIDS AND SECRETIONS 33S

REFERENCES

1 Lopez-Vidriero MT, Das I, Reid L. Bronchorrhea-separation of mucus and serum components in sol and gel phases. Thorax 1979; 34 :512-17 2 Roberts GP. Isolation and characterization of glycoproteins from sputum. Eur J Biochem 1974; 50:265-80 3 Lamblin G, Lhermitte M, Boersma A, Roussel P, Reinhold V. Oligosaccharides of human bronchial glycoproteins. J Bioi Chern 1980; 255:4595-98 4 Clamp JR. Allen A, Gibbons RA, Roberts GP. Chemical aspects of mucus. Br Med Bull 1978; 34:25-41 5 Mossman BT, Craighead JE. Long term maintenance of differentiated respiratory epithelium in organ culture. I. Medium composition. Proc Soc Exp Bioi Med 1975; 149:227-33 6 Gabridge MG. Hamster tracheal organ cultures as models for infection and toxicology studies. Prog Exp Tumor Res 1979; 24:85-95 7 Kaizu T, Lyons SA, Cross CE, Jennings MD, Last JA. Composition of glycoproteins secreted by tracheal explants from various animal species. Comp Biochem Physiol 1979; 62B :195-200 8 Jennings M, Cross CE, Last JA. Glycoprotein synthesis by tracheal explants from various animal species. Comp Biochem Physiol 1977; 57A:317-20 9 Last JA, Jennings MD, Schwartz LW, Cross CE. Glycoprotein secretion by tracheal explants cultured from rats exposed to ozone. Am Rev Respir Dis 1977; 116:695-703 10 Uchida N, Smilowitz H, Tanzer ML. Monovalent ionophores inhibit secretion of procollagen and fibronectin from cultured human fibroblasts. Proc Nat Acad Sci USA 1979; 76: 1868-72 11 Tajiri K, Uchida N, Tanzer ML. Undersulfated proteoglycans are secreted by cultured chondrocytes in the presence of the ionophore monensin. J Bioi Chern 1980; 255 :6036-39 12 Tartakoff A, Vassalli P. Comparative studies of intracellular transport of secretory proteins. J Cell Bioi 1978; 79:694-707 13 Boat TF, Kleinerman Jl, Carlson DM, Maloney WH, Matthews LW. Human respiratory tract secretions. Mucous g)ycoproteins secreted by cultured nasal polyp epithelium from subjects with allergic rhinitis and with cystic fibrosi~ . Am Rev Respir Dis 1974; 110:428-41

Differential Effects of Calcium Ions on Glycoconjugate Secretion by Canine Tracheal Explants* Stephen J. Coles, Ph.D.; John Judge, B.S.; and Lynne Reid, M.D. ecretion of macromolecules from different exocrine glands is known to be dependent upon calcium ions. 1 • 2 In these studies, macromolecule release, induced by various secretagogues, is accompanied by a rise in intracellular calcium originating either from the extra-

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•Department of Pathology Research, Childrens Hospital Medical Center and Harvard Medical School, Boston. Supported by NIH Grant No. HL-22414. Reprint requests: Dr. Coles, Department of Pathowgy, Children's Hospital Medical Center, Boston 02115

34S 24TH ASPEN LUNG CONFERENCE

cellular space or from intracellular organelles. 3 It is not known whether the secretion of mucus by the airway is dependent on calcium ions although, in a short study of the chicken airway, Balfre• demonstrated both CaZ+dependent increases and decreases in mucus secretion. In this study, we have used canine tracheal explants to investigate the effect of calcium ions on both the release of 14 C-glucosamine-labeled glycoconjugates and the activity of airway secretory cells, particularly of the submucosal glands. METHODS

Explants of airway mucosa or full-thickness airway wall were prepared from the anterior region of dog tracheas and incubated, as described previously 5 for 24 hr in 25mM HEPES-buffered medium 199 (Gibco, Grand Island. New York) containing ~Ci/ml 1- 14 C-D-glucosamine. Explants were then washed extensively and reincubated in a modified Earles salts solution (basal medium) composed of 25mM HEPES, 143mM Na +, 5.3mM K +, 1.8mM Ca 2 +, 0.8mM MgZ+, 125mM C1 - , 27mM NO,. - , 0.8mM SO/-, 5.5mM glucose and essential amino acids. All explants were incubated for 30 min in modified Earles medium and then for successive 10 min periods in either modified Earles (to establish baseline glycoconjugate release) or various test media. Media from each period were collected and glycoconjugates precipitated with 10% trichloracetic acid/1% phosphotungstic acid. Precipitates were acid washed and their radioactivity determined by scintillation counting. Normalized labelled glycoconjugate release was calculated as described previously. 5 At the end of each study, explants were fixed in half-strength Karnovsky's fluid and processed for light and e 1ectron microscopy. In certain studies. explants were pulse-labelled with lOO.uCi/ml 3 H-glucosamine and discharge of labelled macromolecules from mucous and serous cells of the submucosal glands quantified by autoradiographic analysis as described earlier. 5

REsuLTS AND DiscussiON Incubation of canine tracheal explants in CaZ+ -free medium + lmM EGTA (CaZ+-free EGTA medium) caused a time-dependent increase in release of labelled glycoconjugates (GC) over a period of at least 90 min (Fig 1). This increase was more marked for mucosal explants (which contained no cartilage) than for explants of full thickness airway wall. In mucosal explants, there· was a "burst" of labeled GC release during the first 20-30 min in CaZ+-free EGTA medium (maximal increase 249% ± 23%; P < 0.001) after which the increase was less marked, stabilizing at 180-200% above baseline after 60 min. In explants of airway wall, incubation in Ca 2 +-free EGTA medium caused a progressive rise in labelled CC release which stabilized at 100120% above baseline after 60 min. Release of labelled GC from explants of tracheal cartilage was not altered in Ca 2 +-free EGTA medium. Histologic examination of explants by light and electron microscopy showed that incubation in Ca 2 +-free EGTA medium caused a progressive exfoliation of surface epithelial cells. Prior removal of the surface epithelium by brushing had no detectable effect either on the degree or time course of the response to Ca 2 +-free EGTA medium indicating

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CHEST, 81: 5, MAY, 1982 SUPPLEMENT