Disruptive effects of anion secretion inhibitors on airway mucus morphology in isolated perfused pig lung

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1 J Physiol (2003), 549.3, pp DOI: /jphysiol The Physiological Society Disruptive effects of anion secretion inhibitors on airway mucus morphology in isolated perfused pig lung Laura Trout, Mary I. Townsley, Amy L. Bowden, and Stephen T. Ballard Department of Physiology, College of Medicine, University of South Alabama, Mobile AL 36688, USA Since anion secretion inhibitors reproduce important aspects of cystic fibrosis (CF) lung disease, the effects of these antagonists on airway mucus morphology were assessed in isolated perfused pig lungs. Maximal inhibitory concentrations of bumetanide and dimethylamiloride, which respectively block Cl _ and HCO 3_ secretion in porcine airways, induced the formation of dense plastered mucus on the airway surface, depletion of periciliary fluid and collapse of cilia. This effect was more pronounced when lungs were also exposed to bethanechol to stimulate submucosal gland secretion, when plastered mucus covered > 98 % of the airway surface. Bethanechol also reduced gland duct mucin content in the absence, but not presence, of the anion secretion inhibitors. Anion secretion inhibitors did not induce measurable increases in goblet cell degranulation. We conclude that inhibition of anion and liquid secretion in porcine lungs disrupts the normal morphology of airway surface mucus, providing further evidence that impaired anion secretion alone could account for critical aspects of CF lung disease. (Received 15 November 2002; accepted after revision 31 March 2003; first published online 17 April 2003) Corresponding author S. T. Ballard: Department of Physiology, MSB 3024, University of South Alabama, Mobile, AL 36688, USA. sballard@usouthal.edu Cystic fibrosis (CF) is a fatal, inherited disease that adversely affects the exocrine function of many organ systems. While severe disruption of pancreatic, intestinal and hepatobiliary secretion occurs in CF, most patients succumb to the pulmonary complications of the disease (Colten, 1991). The earliest pathological changes in the CF lung are obstruction of gland ducts with mucin, which is seen as early as the third trimester of fetal life (Ornoy et al. 1987), and hypertrophy of the submucosal glands (Oppenheimer & Esterly, 1975; Sheppard, 1995). At birth, the lungs of CF patients show no signs of overt disease, but early in childhood, a myriad of pulmonary problems appear which become increasingly severe with age. These complications include severe cough, production of an abnormally thick, viscid mucus, chronic airway infections and a severe impairment of mucociliary transport (Davis, 1993; Regnis et al. 1994). As a consequence of the persistent inflammatory response that accompanies infection, bronchiectasis develops and progresses throughout the life of the patients leading to irreversible loss of pulmonary function (Davis, 1993). Genetic defects in the cystic fibrosis transmembrane conductance regulator protein (CFTR) are the root cause of CF (Riordan et al. 1989). Normally, the CFTR functions as a camp-activated anion channel (Anderson et al. 1991) and, because it is expressed in the apical membrane of airway epithelial cells, can support transepithelial secretion of both Cl _ and HCO 3_ (Smith & Welsh, 1992). While a variety of cellular functions have been attributed to the CFTR, several lines of evidence suggest that this protein is required for normal secretion of liquid by airway epithelia, particularly from submucosal glands, and that loss of this function may be the critical event that leads to the development of CF lung disease. First, CFTR, though present in the airway surface epithelium, is most highly expressed in the serous cells of the submucosal glands (Engelhardt et al. 1992; Jacquot et al. 1993; Ballard et al. 1999). Second, intact submucosal glands and cultured submucosal gland cells from CF airways lose the ability to secrete fluid by a camp-dependent mechanism (Jiang et al. 1997; Joo et al. 2002a). Third, pharmacological blockade of both Cl _ and HCO 3_ secretion in intact bronchi from pigs reproduces many important aspects of CF airway pathology including mucin obstruction of submucosal gland ducts (Inglis et al. 1997a, 1998), production of concentrated airway mucus (Trout et al. 1998b) and impairment of mucociliary transport (Ballard et al. 2002). Unfortunately, the duration of these shortterm in vitro experiments was insufficient to demonstrate whether more chronic manifestations of CF lung disease, such as mucus plugging of distal airways and chronic microbiological infections, are also a consequence of impaired transepithelial anion and liquid secretion. In the present study, we hypothesized that infusion of anion secretion inhibitors through the vasculature of isolated perfused pig lungs could be maintained for prolonged periods which might be sufficient to permit development of more chronic correlates to CF lung

2 846 L. Trout, M. I. Townsley, A. L. Bowden and S. T. Ballard J Physiol pathology. In this study, we observed that inhibition of anion and liquid secretion leads to depletion of periciliary airway liquid, flattening of cilia, and a consequent plastering of mucus to the airway surface. We feel that these observations document the importance of airway anion and liquid secretion to surface mucus morphology and mucociliary transport and could explain the aetiology of important aspects of CF lung disease. METHODS Isolated perfused lung The protocol for animal use was reviewed and approved by the institutional animal care and use committee and complied with US Public Health Service policy on humane care and use of laboratory animals. Young domestic pigs (10 20 kg) were sedated with intramuscular injections of xylazine (4 mg) and ketamine (80 mg). Through an ear vein, intravenous pentobarbital sodium was administered to induce deep anaesthesia and 500 units of heparin were administered to prevent blood coagulation. The right carotid artery was surgically exposed, a cannula inserted and approximately 40 ml of whole blood was collected. The blood was centrifuged, and the plasma was recovered to supplement the perfusion media. The chest was opened and the pulmonary artery and left atrial appendage were cannulated in situ using polyethylene cannulas connected to lengths of silicone tubing. Gravity perfusion of the pulmonary vasculature, in which pressure did not exceed 20 cmh 2 O pressure, was initiated with ice-cold HCO 3_ buffered Krebs-Ringer (KRB)-dextran perfusion solution to flush residual blood from the lung. Then, the trachea, heart and lungs were removed en bloc from the thoracic cavity. The trachea was cannulated approximately 5 mm above the first bronchial branch. The right mainstem bronchus and associated pulmonary vessels were ligated with umbilical tape, and the right lung removed so that only the left lung was ventilated and perfused. The vascular perfusion was then switched from the cold KRB-dextran solution to warm (37 C) plasma-supplemented KRB-dextran solution. To prevent the gradual spontaneous vasoconstriction that is characteristic of isolated lungs from pigs (Gustin et al. 1992), 3 µm isoproterenol was added to the perfusate to reduce vascular smooth muscle tone. We deemed this strategy acceptable in this model since isoproterenol has been shown to have no measurable stimulatory effect on liquid secretion in porcine bronchi (Trout et al. 2001). The lungs were perfused with a Masterflex roller pump and the flow rate was adjusted to maintain a mean pulmonary artery pressure of 12.0 ± 0.3 cmh 2 O (n = 22). Flow rates in all experiments were 550 ± 22 ml min _1 (100 g lung wet weight) _1 (n = 22). Venous pressure, controlled by adjusting the height of the perfusion reservoir, was maintained at 5 cmh 2 O (relative to the hilus) except during periods when it was elevated to measure vascular permeability. The perfusate ph was maintained at 7.4 by ventilating the lungs with humidified gas (30 % O 2 5 % CO 2 65 % N 2 ) at tidal volumes of ml, ventilation rates of breaths min _1, and 3 cmh 2 O positive end expiratory pressure (PEEP). To monitor the stability of the preparation, the capillary filtration coefficient (K f,c ) was measured at the beginning and end of every experiment. To measure the K f,c, venous pressure was raised from 5 to cmh 2 O for 15 min. K f,c was calculated using the following equation: K f,c =(DW/Dt)/DP c, where DW/Dt is the slope of the steady-state weight-gain curve measured during the capillary filtration phase between min 13 and 15 following venous pressure elevation (Townsley et al. 1995), and DP c is the change in capillary pressure measured before and at the end of the increased venous pressure period. DW/Dt was corrected for any non-isogravimetric state prior to the K f,c manoeuvre. P c was measured using the double occlusion technique (Townsley et al. 1986). K f,c was expressed in ml min _1 cmh 2 O _1 (100 g lung initial wet weight) _1. At the end of the 15 min period, the venous pressure was returned to 5 cmh 2 O and a 15 min recovery period was allowed for the re-equilibration of vascular filtration. Total vascular resistance (R t ) was calculated as the difference in arterial and venous pressures divided by the vascular flow rate. Arterial and venous pressures were taken just prior to K f,c measurements. R t is expressed in cmh 2 Ol _1 min _1 (100 g lung initial wet weight) _1. Following recovery from the initial K f,c measurement, the combination of bumetanide (100 µm) and dimethylamiloride (DMA, 100 µm) or the drug vehicle dimethyl sulfoxide (DMSO) was added to the vascular perfusate. The combination of these inhibitors has been shown to block ~90 % of acetylcholine- or substance P-induced liquid secretion in porcine bronchi by respectively inhibiting Cl _ and HCO _ 3 secretion (Ballard et al. 1999; Trout et al. 2001). A period of 45 min was allowed for the inhibitors to take effect. Then bethanechol, a non-metabolizable muscarinic agonist (or an equal volume of its vehicle, water) was added to the perfusate to stimulate airway liquid secretion. Four hours after adding the inhibitors, a final K f,c measurement was made followed by a 15 min recovery period. The lung was then perfusion fixed for 20 min with glutaraldehyde fixative at peak inflation volume. The lung was removed from the cannulas, and two adjacent tissue blocks (each transecting the central bronchus) were taken from the mid-portion of the lower lobe. To preserve the airway surface mucus features, one of the two blocks was placed in osmium and/or perfluorocarbon fixative solution for 1.5 h and then transferred to 100 % ethanol. Tissue blocks fixed only by glutaraldehyde perfusion were processed for light microscopy by conventional methods and stained for mucin with alcian blue and pyronin Y (ph 2.5). Tissue blocks that were subsequently fixed with osmium were embedded in plastic and processed for light and transmission electron microscopy (TEM) by conventional methods. Sections of the osmium-fixed airways that were processed for light microscopy were stained with toludine blue. Excised bronchi To selectively assess the possible effect of isoproterenol on gland duct mucin content, subsegmental bronchi of approximately 2 4 mm diameter were dissected from lungs of pigs immediately following euthanasia. Paired bronchi were warmed to 37 C in Krebs buffer and received either no treatment (water vehicle) or 3 µm isoproterenol. After 5.5 h exposure (a time period equal to that of the isolated lung preparation), the airways were removed from the treatment solutions and immediately placed in glutaraldehyde fixative and subsequently processed for light microscopy and stained for mucins with alcian blue and pyronin Y as described above. This method has been successfully used in past studies to document conditions which cause mucin obstruction of gland ducts (Inglis et al. 1997a, 1998). Morphometric analyses From the alcian blue and pyronin Y-stained slide sections, the relative quantity of mucin within the gland ducts was subjectively

3 J Physiol Disruptive effects of anion secretion 847 judged. In a blinded fashion, each gland duct in a slide section received a graded score between 0 (no visible mucus present) and 5 (complete mucin occlusion of the gland duct). Three slide sections, taken from different areas of the same airway, were scored and averaged. The frequency distribution of individual scores was tabulated for each drug treatment. Some gland ducts were impossible to score for mucin content because the duct lumen was not visible. This could have been due to chance, (i.e. sectioning of some ducts at oblique angles) or ducts could have collapsed due to reduced luminal volume. These ducts were also counted. The fraction of airway surface covered with dense plastered mucus was measured from representative osmium-fixed slide sections. Digital images of the sections were obtained with a Zeiss ACM microscope equipped with a Nikon Coolpix digital camera. A collage of each airway section was generated by assembling serial digital images using Corel PhotoPaint 8 software. Airway surface dimensions were measured from the collage images with Scion Image 4.0.2, a PC version of the NIH image analysis program. Goblet cell secretory ratio was determined by the method of Tokuyama and coworkers (1990). All goblet cells in the tissue sections were blindly graded according to the amount of intracellular mucin that was present. Goblet cells were scored as either grade 1 (cells that had discharged their mucin, judged as the vertical distance of the stained area being contained within the apical one-third of the total thickness of the epithelial layer) or grade 2 (cells that had not discharged their mucin, judged as the vertical distance of the stained area exceeding the apical one-third of epithelial layer). The secretory ratio, which should vary according to goblet cell discharge rate, was calculated as the ratio of the total grade 1 and grade 2 mucin scores. Solutions and drugs Bicarbonate-buffered Krebs-Ringer solution (KRB) contained (mm): NaCl, 4.7 KCl, 2.5 CaCl 2, 2.4 MgSO 4, 1.2 KH 2 PO 4, 25.0 NaHCO 3, and 11.6 glucose. Perfusion solutions consisted of KRB with 6 % dextran and 3 % autologous plasma. The initial cold KRB perfusion solution contained dextran but no plasma. Aqueous fixation solution was prepared by dissolving paraformaldehyde (1 % final concentration) in hot (60 C) Millonig s buffer (composed of 136 mm NaH 2 PO 4 and 107 mm NaOH in water) under a fume hood. After cooling, the ph was adjusted to 7.2 if necessary. On the day of use, glutaraldehyde (1 % final concentration) and cetylpyridinium chloride (0.025 % final concentration) were added to the solutions. Osmium/ perfluorocarbon fixation solution was prepared by dissolving OsO 4 (1 % final concentration) in Fluorinert FC-72 (SynQuest Labs, Alachua, FL) overnight under a fume hood. Statistical analyses Comparisons of groups where paired values were obtained in the same tissues (i.e. initial and final K f,c and vascular resistances) were made using Student s paired t tests. Other groups were compared using ANOVA. Differences were considered significant when P < Data are presented as means ± S.E.M. The number of observations (i.e. number of lungs, each from a different animal) is represented as n. RESULTS To monitor the haemodynamic stability of the perfused lungs, K f,c and segmental vascular resistances were measured at the beginning and end of each experiment. The baseline K f,c values, though somewhat variable, were not statistically different between treatment groups (Table 1). The K f,c averaged ± ml min _1 cmh 2 O _1 (100 g) _1 (n = 22) overall at baseline, indicative of normal vascular permeability. These data are similar to Figure 1. Effect of anion secretion inhibitors and bethanechol on gland duct mucin content Ordinates indicate the frequency (fraction of the total) of gland ducts which received graded mucin content scores between 0 (no visible mucin) and 5 (gland duct completely occluded with mucin). Charts on the right side received bethanechol while those shown on the left side did not. The average number of ducts counted for each lung was ± 20.1 (vehicle only, n = 6), ± 37.6 (bethanechol only, n = 5), 68.0 ± 21.4 (bumetanide + DMA only, n = 5), and ± 15.9 (bumetanide + DMA + bethanechol, n = 6). * Significant differences from untreated airways without bethanechol. Significant difference from untreated airways with bethanechol.

4 848 L. Trout, M. I. Townsley, A. L. Bowden and S. T. Ballard J Physiol those previously reported for pig and dog lung (Townsley et al. 1995; Fadel et al. 1998). By the end of the perfusion experiments, K f,c increased significantly from baseline only following bumetanide + DMA pretreatment without bethanechol, but K f,c nonetheless remained within normal values. Final average K f,c was higher when bethanechol was added following bumetanide + DMA pretreatment, but this change was principally due to a single elevated value (0.208 ml min _1 cmh 2 O _1 (100 g) _1 ) and the means were not significantly different. Baseline total vascular resistances were substantially less than reported by Gustin et al. (1992) and were not significantly affected by inhibitor or secretagogue treatment (Table 2). Together, these data indicate that the isolated, perfused pig lung preparation remains stable over the time course of these experiments. Relative mucin content in submucosal gland ducts, assessed by blinded subjective scoring of slide sections, is shown in Fig. 1. In the absence of the glandular secretagogue bethanechol, gland ducts contained relatively large amounts of mucin with both inhibitor and vehicle pretreatments, as evidenced by the high mean frequency (> 0.40) of maximum mucin scores. In the presence of bethanechol, the mucin score frequency Figure 2. Effect of anion secretion inhibitors and bethanechol on gland duct patency The ordinate shows the frequency (as fractions of the total) of gland ducts that had no visible lumen in slide sections. The average number of non-visible ducts counted for each lung was 25.2 ± 8.4 (vehicle only), 10.7 ± 4.4 (bethanechol only), 67.3 ± 22.8 (bumetanide + DMA only), and 15.2 ± 3.4 (bumetanide + DMA + bethanechol). Significant difference from no pretreatment without bethanechol. * Significant difference from bumetanide+dma pretreatment without bethanechol. Figure 3. Effect of isoproterenol on gland duct mucin content in excised porcine bronchi Ordinates indicate the frequency (fraction of the total) of gland ducts which received graded mucin content scores between 0 (no visible mucin) and 5 (gland duct completely occluded with mucin). Untreated bronchi received only water vehicle while treated airways received 3 µm isoproterenol. Incubation time was 5.5 h, comparable to isolated lung exposure. The average number of ducts counted for each lung was 389 ± 44 (untreated, n = 5) and 314 ± 76 (isoproterenol, n = 5). No significant differences in mucin scores were observed with isoproterenol treatment.

5 J Physiol Disruptive effects of anion secretion 849 distributions became noticeably skewed to the left in lungs pretreated with only vehicle indicating that mucin content of the ducts was decreased. Indeed, the most frequent mucin score under these conditions was 0, indicating no visible mucin in the gland ducts. These data are consistent with induction of liquid secretion in glands by bethanechol. Lungs that had been pretreated with bumetanide + DMA, however, maintained the relatively high ductal mucin content in the presence of bethanechol where the mucin score with the greatest frequency was still 5. The frequency of non-visible duct lumena was significantly higher in lungs treated with only bumetanide + DMA than untreated controls (Fig. 2). However, in all lungs that received bethanechol treatment, the frequency of non-visible ducts was low and comparable with untreated lungs. The difference between bethanechol and vehicle treatment in bumetanide + DMApretreated lungs was significant. These results suggest that bumetanide + DMA treatment reduced the secretion volume within gland ducts. Further, bethanechol appears to increase gland duct volume in bumetanide + DMApretreated lungs with the ducts in this group being distended and containing relatively high amounts of mucin. These findings are consistent with the notion that bumetanide + DMA blocks the majority of the liquid but not the mucin secretion response to cholinergic agonists. The relatively high gland duct mucin content in the control lungs was unexpected since previous studies of excised airways showed that gland ducts from untreated airways contained little mucin (Inglis et al. 1997a). We reasoned that this finding may have been related to the presence of isoproterenol which could have induced small quantities of mucin-rich gland secretions that could not be detected in a previous study of glandular liquid secretagogues (Trout et al. 2001). To test this notion, the effect of isoproterenol on gland duct mucin content was assessed in excised porcine bronchi. As seen in Fig. 3, gland ducts from airways exposed to isoproterenol as well as airways that received no treatment exhibited no skewdness of the mucin distribution curves suggesting that isoproterenol alone was not responsible for the relatively high scores in the untreated isolated perfused lungs. Figure 4. Light micrographs of intralobular bronchi: effect of anion secretion inhibitors Top, representative bronchus from lung exposed to bethanechol only. Cilia on the airway surface appear normal and there is little or no mucus present. Bottom, representative bronchus from lung pretreated with bumetanide + DMA and then exposed to bethanechol. Note that the airway appears normal except that cilia are not visible and the airway surface is covered with a thin layer of dense mucus. Scale bars 50 µm. Figure 5. Transmission electron micrographs (TEM) of intralobular bronchi: effect of anion secretion inhibitors Top, representative bronchus from lung exposed to bethanechol only. Cilia morphology appears normal. Bottom, representative bronchus from lung pretreated with bumetanide + DMA and then exposed to bethanechol. Note that the cilia are collapsed between the dense, overlying mucus and the epithelial cell surface. Scale bars 5 µm.

6 850 L. Trout, M. I. Townsley, A. L. Bowden and S. T. Ballard J Physiol In osmium-fixed airways that had been pretreated with bumetanide + DMA and then exposed to bethanechol, the surface epithelium was covered with a thin, dense layer of mucus that appeared to be plastered to the airway surface such that the cilia were not visible in light micrographs (Fig. 4). TEM of the mucosal surface of these cells shows that the cilia are completely flattened under this layer of dense mucus (Fig. 5). In contrast, airways exposed only to bethanachol exhibited normal surface morphology with prominent cilia and little, if any, detectable mucus (Figs 4 and 5). From the osmium-fixed sections, the prevalence of this plastered mucus in the airways of the lungs was determined by expressing the fraction of the airway surface covered in this manner to the total airway surface in each section (Fig. 6). In the absence of bethanechol and inhibitors, only 21.6 ± 10.8 % of the airway surface was covered with the plastered mucus. When lungs were exposed to bethanechol only, no plastered mucus was observed in any lung sections. In contrast, when lungs were pretreated with only bumetanide + DMA, 61.2 ± 19.8 % of the airway surface was covered with plastered mucus, a value significantly different from that in untreated lungs. But, when bumetanide + DMA- pretreated lungs were also exposed to bethanechol, 98.0 ± 2.0 % of the surface became covered with the plastered mucus. A likely explanation for these findings is that stimulation of gland secretion normally supplies enough liquid to the airway surface to permit clearance of mucus by mucociliary transport. However, when liquid secretion from glands is blocked, stimulation of glands results in secretion of dense mucus which accumulates on the surface due to depletion of periciliary liquid and impairment of mucociliary transport. In some slide sections from bumetanide + DMA-treated lungs, evidence of goblet cell discharge into the plastered mucus layer could be seen (Fig. 7). To determine whether goblet cell mucin secretion increased under these conditions, the secretory ratio of goblet cells was measured in the stained slide sections. While the variance associated with some treatments was large, no significant difference in secretory ratio was observed (Fig. 8) suggesting that there was no consistent measurable change in the rate of goblet cell discharge among the various treatments in this study. DISCUSSION Airway mucus liquid normally exists in both gel and sol phases (Wu et al. 1998). The mucin-rich gel phase lies atop the cilia and is swept towards the cranial end by the beating action of the cilia. The sol phase, which lies beneath the mucus gel, is the low viscocity fluid through which the cilia beat and whose depth is normally approximated by the length of the outstretched cilia. This periciliary liquid layer at the airway surface is maintained by a balance of liquid Figure 6. Effect of anion secretion inhibitors and bethanechol on the presence of plastered surface mucus Ordinate shows the percentage of the airway surface covered with dense mucus (see lower panel, Fig. 3). * Significant difference from lungs in the same pretreatment group that did not receive bethanechol. Significant difference from lungs that received no inhibitor pretreatment. Figure 7. Light micrographs of intralobular bronchi: effect of anion secretion inhibitors on mucin discharge from goblet cells Top, representative bronchus from lung exposed to bethanechol only. Note the presence of mucin granules in goblet cells and the absence of mucus on the airway surface. Bottom, representative bronchus from lung pretreated with bumetanide + DMA and then exposed to bethanechol. Note mucins that have been discharged from goblet cells are trapped in the mucus layer directly above the epithelium. Scale bars 20 µm.

7 J Physiol Disruptive effects of anion secretion 851 secretion and liquid absorption (Wu et al. 1998) and is critical to mucociliary transport function. In the present study, we demonstrate that inhibition of transepithelial anion secretion in the isolated lungs of pigs disrupts the normal morphology of airway surface mucus causing depletion of the periciliary liquid layer and flattening of the cilia between the overlying mucus layer and the surface epithelial cells. This effect was most striking when bethanechol was present to maximally stimulate submucosal gland secretion. Depletion of this periciliary liquid layer is the probable cause of the mucociliary stasis induced by these same anion secretion inhibitors in a previous study (Ballard et al. 2002). These results have important relevance to CF lung pathogenesis. Scanning electron micrographs (SEM) of the surfaces of CF airways similarly show the presence of a thick adherent mucus that flattened and became matted with the cilia (Simel et al. 1984), paralleling observations in the present study. Depletion of the periciliary fluid layer and mucus adherence to ciliated cells has also been documented in CF cultured airway epithelia (Knowles & Boucher, 2002). These observations probably account for the defect in mucociliary transport which characterizes this disease. The SEM study also noted that the gland ducts were unusually dilated and obstructed with thick mucus strands (Simel et al. 1984), a phenomenon that has also been reproduced in porcine excised bronchi exposed to bumetanide and DMA (Inglis et al. 1997a, 1998). Consequently, our findings document that critical aspects of CF lung pathology can be directly attributed to impaired anion secretion. Both the airway surface epithelium and the submucosal glands possess the capability to secrete anions and liquid but the relative contribution of these two regions to normal maintenance of this surface liquid layer is unclear. Undoubtedly, anion and liquid secretion occur from submucosal glands of the tracheobronchial airways, the magnitudes of which depend upon the level of stimulation. Even in the absence of stimulation, submucosal glands appear to secrete liquid at a relatively low rate (Ueki et al. 1980; Inglis et al. 1997b; Joo et al. 2002b). However, cholinergic or tachykinin stimulation induces copious secretion of fluid from glands (Quinton, 1979; Haxhiu et al. 1990; Ballard et al. 1999; Trout et al. 2001; Joo et al. 2002b) which is generated by a combination of active Cl _ and HCO 3_ secretion ( Trout et al. 1998a, 2001; Ballard et al. 1999). When Cl _ and HCO 3 _ secretion are inhibited with either maximally effective concentrations of NPPB or the combination of bumetanide + DMA, glandular liquid secretion is reduced by ~90 % (Ballard et al. 1999; Trout et al. 2001). Comparatively less evidence exists supporting a role for anion and liquid secretion by surface epithelium, though logically this barrier must contribute to airway surface liquid since some species, such as mice and rabbits, exhibit few if any submucosal glands. Using cultured human bronchial epithelium, Tarran and coworkers reported evidence that the ratio of Cl _ secretion to Na + absorption increases as the depth of airway surface liquid decreases, suggesting that fluid secretion by these cells may be induced only when luminal fluid is depleted (Tarran et al. 2001). In the present study, we used inhibitors that were previously shown to block glandular liquid secretion, but we cannot discount the possibility that the same agents inhibit secretion of small but important quantities of liquid from the surface epithelium as well. Regardless of the site of action, however, our findings document the importance of anion secretion to the maintenance of normal airway mucus and periciliary liquid. Depletion of periciliary airway liquid in the presence of the anion secretion inhibitors probably occurs as the consequence of ongoing ENaC-dependent absorption of Na +. This conclusion can be reached from at least two observations. First, inhibition of Cl _ and HCO 3_ secretion has been shown to substantially reduce mucociliary transport in pig tracheas, a response that is significantly attenuated when absorption of luminal liquid is also inhibited by the ENaC blocker benzamil (Ballard et al. 2002). Second, ENaC-dependent Na + absorption has been demonstrated to not only persist in CF but to be elevated above normal levels (Boucher et al. 1986). This later observation has led to several clinical trials using inhibitors of ENaC as treatments for CF with mixed results (Knowles et al. 1990; Bowler et al. 1995; Pons et al. 2000). Our findings suggest that inhibition of anion secretion alone, while maintaining presumably normal rates of Na + absorption, is capable of causing periciliary fluid depletion, generation of plastered surface mucus and Figure 8. Effect of anion secretion inhibitors and bethanechol on goblet cell secretory ratio Ordinate shows the goblet cell secretory ratio for the indicated drug treatments. Mean numbers of total cells counted from each lung: no pretreatment = ± 60.0, bethanechol only = ± 78.7, bumetanide+dma only = ± 80.3, bumetanide+dma+bethanechol = ± No significant differences were observed with any treatment.

8 852 L. Trout, M. I. Townsley, A. L. Bowden and S. T. Ballard J Physiol reduced mucociliary transport. We believe therefore that manoeuvres which are designed to increase delivery of liquid to the airway surface, by either endogenous or exogenous routes, are likely to be beneficial adjuncts to CF therapies. In the present study, we observed that, on average, gland ducts contained relatively large quantities of mucin in the absence of anion secretion inhibitors and bethanechol. This finding is at odds with previous studies of excised pig bronchi where untreated tissues exhibited relatively low ductal mucin content (Inglis et al. 1997a). This result was unexpected but could possibly have resulted from the presence of isoproterenol, which was added to maintain low vascular resistance. We reasoned that this agent would be a good choice for this purpose since isoproterenol has been previously shown to have no significant effect on the volume secretion from porcine bronchi (Trout et al. 2001; Joo et al. 2002b) or feline submucosal glands (Quinton, 1979). However, this agent could have induced a low level of mucin secretion resulting in observable ductal mucin accumulation without significantly affecting the volume secretion. When we exposed excised porcine bronchi to isoproterenol, gland duct mucin scores did not exhibit the rightward skewdness seen in the control bronchi from the isolated lungs, suggesting that this agent was probably not responsible for this observation. We must therefore assume that the relatively high ductal mucin scores in the untreated airways in the present study was due to some aspect of the isolated perfused lung technique. One possibility is that the interruption of the bronchial circulation, an unavoidable consequence of isolating the lungs, may have reduced the capacity for the airways to secrete liquid. We think that this is unlikely for the following reasons. While the source of normal blood flow to the airway circulation, i.e. the bronchial artery, is severed during lung excision, the bronchial vessels should receive retrograde perfusion from the pulmonary circulation, the normal termination point for these vessels. Evidence that the airways are not volume starved can be drawn from the responses to bethanechol alone, which reduced gland duct mucin content and the plastered surface mucus, findings consistent with liquid volume secretion. Consequently, we cannot account from available information for the relatively high mucin content of the control gland ducts. We observed in some light micrographs that the discharged mucin contents of goblet cells were visible in the plastered mucus layer directly above the cells (Fig. 6). This observation suggests: (1) mucociliary transport could have been impaired in these tissues and/or (2) the presence of this plastered mucus on the airway surface, or the inhibitors themselves, could have induced goblet cell secretion. The first possibility has been confirmed in a previous study, where the same anion secretion inhibitors substantially reduced mucociliary transport in porcine tracheas (Ballard et al. 2002). To address the latter possibility, we measured the secretory ratio of the goblet cells (i.e. the fraction of goblet cells that were depleted of apical mucin) as described by Tokuyama and coworkers (1990). We saw no significant difference in this parameter between any of the treatment groups. 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This work was funded by grants from the US National Institutes of Health (HL and HL 48622) as well as a Cystic Fibrosis Foundation Pilot and Feasibility Grant (BALLAR99I0). Supplementary material The online version of this paper can be found at: DOI: /jphysiol and contains supplementary material entitled: Collages of osmium/perfluorocarbon-fixed airway sections from isolated lungs It consists of two figures, representative collages of airway sections either treated with bethanechol (Supplemental Fig. 1) or pretreated with bumetanide + DMA and subsequently treated with bethanechol (Supplemental Fig. 2).