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1Department of Physiology, College of Medicine, University of South Alabama, Mobile, Alabama; and 2Maternal and Child Health Sciences, Ninewells Hospital, Dundee University, Dundee, United Kingdom
Submitted 11 April 2005 ; accepted in final form 4 August 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONAPPENDIXGRANTSREFERENCES |
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cystic fibrosis; airway liquid secretion; submucosal glands
-adrenergic stimulation is also capable of inducing significant liquid secretion (21, 23). Although the ionic mechanism responsible for elaboration of this fluid remains to be fully defined, there is little doubt that gland liquid is largely generated through the active transepithelial secretion of anions (3, 29, 30). Several lines of evidence suggests that the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated anion channel, plays an integral role in the secretion of gland liquid. First, CFTR has been shown to be heavily expressed in the apical membrane of gland serous cells by immunohistochemistry and in situ hybridization (7, 11), although these findings have been recently challenged (17). Second, numerous pharmacological inhibitors of the CFTR have been shown to be efficacious inhibitors of gland liquid secretion (3, 28, 29). Third, and most convincingly, airway submucosal glands from the lungs of human CF patients, who express genetically defective CFTR, lose the ability to respond to either vasoactive intestinal peptide (VIP) or forskolin, agents that induce secretion in normal airways by elevating intracellular levels of cAMP (13). These findings suggest that a defect in liquid secretion from glands may underlie the complex pulmonary pathology of this disease, which includes production of abnormally thick inspissated mucus, impairment of mucociliary and cough clearance, and chronic airway infections. Unfortunately, the ionic mechanism that underlies cAMP-dependent liquid secretion from glands is not completely understood. The objective of the present study was therefore to better define the mechanism responsible for cAMP-activated ion and liquid transport pathways in airway glands.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONAPPENDIXGRANTSREFERENCES |
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10–15 kg) were sedated with intramuscular xylazine (4 mg) and ketamine (80 mg) and killed with an intravenous overdose of pentobarbital sodium. The lung lobes were removed and placed in Krebs Ringer bicarbonate (KRB) at room temperature. Segments of intralobular bronchi (30–40 mm in length) were dissected from the lung parenchyma, and the side branches of the airways were ligated with suture. Bronchi were slowly warmed to 37°C (0.1°C/min) in a KRB bath. At the end of the warming period, the airways were exposed to a variety of pretreatments designed to inhibit specific transport processes. One or more of the following transport inhibitors were added to the KRB bath. Bumetanide (10 µM), a loop diuretic that inhibits Na+-K+-2Cl– cotransport (NKCC), was used to block transepithelial Cl– secretion. Dimethylamiloride (DMA, 100 µM), an inhibitor of Na+/H+ exchange (NHE), was used to block extrusion of H+ across the basolateral membrane, a maneuver that effectively inhibits HCO3– secretion that is dependent on intracellular HCO3– generation (27). 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 1 mM) was used as a potential inhibitor of Cl–/ HCO3– exchange (AE), Na+-nHCO3– cotransport (NBC), Na+-dependent Cl–/HCO3– exchange (NDAE), or possibly non-CFTR anion channels. 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 300 µM), diphenylamine-2-carboxylate (DPC, 1 mM), and glibenclamide (1 mM), which inhibit a number of different anion channels including CFTR (25), were used as anion channel blockers. The effect of niflumic acid (1 mM), which has often been used to block Ca2+-activated anion channels (12, 15, 16) and recently shown to exhibit CFTR blocking activity as well (26), was also assessed. Acetazolamide (10 mM) was used to inhibit carbonic anhydrase and thereby reduce HCO3– synthesis. In some experiments, the normal KRB bath solution was replaced with equal volumes of warm (37°C) Cl–-free KRB, HCO3–-free KRB, Na+-free KRB, or Ca2+-free KRB. During this incubation period, the ends of the airways were open to the bath solution, and the inhibitors had access to both the luminal and adventitial sides of the tissues.
After a 45-min incubation period with the above pretreatments, the airways were removed from their bath solutions, and all accessible fluid and mucus was removed from their lumens. The bronchi were then tied with suture onto polyethylene cannulas and returned to their respective baths. Forskolin (10 µM), a direct activator of adenylyl cyclase, was added to the bath to elevate intracellular cAMP and stimulate anion and liquid secretion. All inhibitor experiments were performed with paired control airways that were exposed to only forskolin and the inhibitor vehicle, dimethyl sulfoxide (DMSO).
After a 2-h incubation with forskolin, the bronchi were removed from their cannulas and sectioned lengthwise, and all accessible mucus liquid was recovered from the lumens. Mucus liquid was placed into tared tubes, sealed, and then weighed to determine secretion volume. This method was deemed appropriate to assess liquid flux since the nonvolatile solids content of the secreted liquid is unlikely to exceed 5% of the liquid weight under these conditions (31). Liquid samples were frozen and stored at –70°C for later analysis. Airway lengths and outer diameters were measured and used to estimate luminal surface areas as previously described (30). Net liquid secretion rates (Jv) were calculated based on the total secretion volume, the luminal surface area, and the time of secretagogue exposure.
Removal of the surface epithelium. To determine whether the source of the secreted liquid was the surface epithelium or the submucosal glands, the surface epithelium of some airways was abrasively removed with a wooden ream, and the tissues were exposed to forskolin with and without a secretion inhibitor. The mucosal surface of selected airways was examined with a Zeiss ACM microscope equipped with a x20 water immersion objective to confirm that this technique adequately removed the surface epithelium and left the submucosal structures intact.
Solution instillation. To assess the magnitude of possible temporal changes in HCO3– concentration in the luminal liquid due to diffusion or transport across the surface epithelium, solutions of varying HCO3– composition were instilled into the lumen of cannulated bronchi. These solutions consisted of either normal KRB (25 mM HCO3–), 150 mM NaCl (HCO3–-free), or 150 mM NaHCO3. The submucosal bath was always normal KRB (25 mM HCO3–). Otherwise, the airways were not exposed to any inhibitors, vehicle, or secretagogues. After 2-h incubation with the instillates, the airways were removed from the cannulas, and the residual luminal solutions were collected, frozen, and later assayed for HCO3– concentration.
Bicarbonate analysis. Frozen samples were thawed, and HCO3– concentrations were determined with an Infinity CO2 kit (Sigma Chemical/Thermo Electron). Because the HCO3– concentrations in liquid from bumetanide-treated tissues were typically higher than control treatments, these samples were diluted to ensure that the concentrations would fall within the linear range of the assay.
Solution composition and drugs. Normal KRB contained 112.0 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 2.4 mM MgSO4, 1.2 mM KH2PO4, 25.0 mM NaHCO3, and 11.6 mM glucose. For Cl–-free KRB, NaCl and KCl were respectively replaced with equimolar Na-gluconate and K-gluconate. To compensate for the Ca2+ chelating effects of gluconate ions, 4.5 mM Ca-gluconate was added to the solution (19). For Na+-free KRB, NaCl and NaHCO3 were respectively replaced with equimolar choline-Cl and choline-HCO3. To make Ca2+-free KRB, CaCl2 was omitted from the solution and 2.5 mM EGTA was added to chelate free Ca2+. For HCO3–-free KRB, NaHCO3 was replaced with 25 mM HEPES buffer, and the solution was titrated to pH 7.4. High-HCO3– solution was composed of only 150 mM NaHCO3. When HCO3–-free KRB was used, the solution was gassed with 100% O2. Otherwise, the pH of all KRB solutions was maintained at 7.4 by constant gassing with 95% O2/5% CO2. NPPB was obtained from Calbiochem. DPC was purchased from Aldrich as N-phenylanthranillic acid. All other drugs were purchased from Sigma Chemical. Stock solutions of all inhibitors and forskolin were prepared with DMSO.
Statistics. Data are reported as means ± SE. The number of bronchi in each group are represented by n with each tissue taken from a different animal. The only exception to this is when aggregate forskolin control statistics are reported for liquid secretion rates and HCO3– concentrations where responses from two control bronchi, used for comparing two different inhibitor treatments, were sometimes taken from the same animal. All inhibitor experiments were performed with paired control airways that were exposed to only the inhibitor vehicle and forskolin. Paired comparisons were made with a paired Student’s t-test, and unpaired comparisons were made by ANOVA (SigmaStat software, version 2.0). P < 0.05 was considered significant. All statistical comparisons were made on the raw data. However, for clarity, much of the data are expressed in the figures as percentages of their respective control responses.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONAPPENDIXGRANTSREFERENCES |
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Ion replacement. The Jv response to forskolin was profoundly reduced when tissues were exposed to Na+-free solution (6.8 ± 1.8% of control, n = 6), indicating that virtually all secretory processes were dependent upon extracellular Na+ (Fig. 1A). The Jv was significantly reduced by approximately one-half in tissues bathed in Cl–-free solution (46.9 ± 7.2% of control, n = 6) and HCO3–-free solution (56.3 ± 14.6% of control, n = 7), suggesting prominent requirements for these ions as well. Removal of Ca2+ from the bath solution also significantly reduced the Jv response (37.8 ± 9.3% of control, n = 7) in these tissues; thus Ca2+ signaling likely plays an important role in the secretion response to forskolin.
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Anion channel blockers. The effects of a number of potential anion channel inhibitors were assessed on forskolin-induced secretion (Fig. 2A). The CFTR inhibitors NPPB, DPC, and glibenclamide all significantly reduced the Jv response to forskolin [12.6 ± 6.4% of control (n = 12), 18.8 ± 5.8% of control (n = 8) and 37.1 ± 10.3% of control (n = 7), respectively]. Niflumic acid, a more potent inhibitor of Ca2+-activated anion channels than CFTR (26), had no significant effect on the forskolin-induced JV in these airways (86.1 ± 11.8% of control, n = 6). Inhibition of the Jv by NPPB, DPC, and glibenclamide would be consistent with CFTR participation in forskolin-induced liquid secretion, whereas lack of a niflumic acid response would not. DIDS, which inhibits numerous non-CFTR anion channels as well as several anion transport proteins, significantly reduced the Jv to 55.3 ± 10.8% of control (n = 6). A partial inhibitory effect of DIDS suggests that either an alternate anion channel or one of several possible anion exchangers and/or cotransporters participates in the secretion response.
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Bicarbonate secretion inhibitors. We reasoned that intracellular HCO3– generation could be facilitated by carbonic anhydrase and removal of H+ from the cytoplasm by basolateral NHE. When acetazolamide, an inhibitor of carbonic anhydrase, was applied to the airways, no effect on forskolin-induced Jv was seen (94.9 ± 24.4% of control, n = 5) (Fig. 3A). Similarly, no significant effect on Jv was seen with DMA, an inhibitor of NHE (75.9 ± 11.2% of control, n = 6). When DIDS was combined with DMA, Jv was significantly inhibited to a similar level as seen with DIDS alone (60.5 ± 9.4% of control, n = 7). Taken alone, these data suggest that forskolin-induced volume secretion is insensitive to carbonic anhydrase or NHE inhibition.
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Bumetanide and bicarbonate secretion inhibitors. Bumetanide had no significant effect on the forskolin-induced Jv (84.6 ± 13.7% of control, n = 7) (Fig. 4A). This was surprising given that about one-half of the Jv was dependent on bath Cl– (see Fig. 1A). A number of potential HCO3– secretion inhibitors were thus used in combination with bumetanide to determine whether the bumetanide-insensitive Jv was due to secretion of this anion. Addition of either DMA or DIDS to the bumetanide pretreatment significantly reduced the forskolin-induced Jv to 52.6 ± 4.4% (n = 6) and 52.0 ± 5.9% (n = 8) of their respective controls. The combination of bumetanide, DMA, and DIDS essentially abolished the Jv response to forskolin (8.6 ± 2.8% of control, n = 6), as did bumetanide in HCO3–-free solution (11.7 ± 2.6% of control, n = 6). Although it had no effect of its own on the forskolin-induced Jv (see Fig. 3A), acetazolamide substantially reduced the Jv to 24.0 ± 6.6% of control (n = 7) in the presence of bumetanide (Fig. 4A). Accordingly, DMA-inhibitable HCO3– secretion appeared to have been induced by bumetanide since no significant effect of DMA was seen in the absence of this loop diuretic. The fractional inhibitory effects of DIDS on HCO3– secretion persist in the presence of bumetanide such that the combination of bumetanide, DMA, and DIDS inhibits virtually all of the forskolin-induced Jv. Abolition of the Jv by acetazolamide in the presence but not absence of bumetanide supports our speculation (see Bicarbonate secretion inhibitors) that carbonic anhydrase inhibition by itself induces Cl– secretion in these tissues. Furthermore, the observation that Cl–-free solution blocks nearly all of the Jv in the presence of DIDS (21.9 ± 2.7% of control, n = 6) (Fig. 4A) substantiates the notion that the DMA-sensitive component of the Jv is inducible by bumetanide, whereas the DIDS-sensitive component is not.
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Removal of the surface epithelium. To localize the site of forskolin-induced liquid secretion, forskolin was applied to some airways whose surface epithelium was abrasively removed leaving the submucosal glands intact. Epithelium removal had no significant effect on the Jv response to forskolin or to its inhibition by NPPB (Fig. 5). Consequently, the liquid secretion response to forskolin was likely to have originated from submucosal glands.
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150 mM). However, in the tissues pretreated with bumetanide, the HCO3– concentration in the secreted liquid was not higher than 41.9 meq/l. We reasoned that high concentrations of HCO3– may not have been sustainable across the surface epithelium because of the potential for this anion to leak across the tight junctions into the submucosal bath where the concentration was 25 mM. To test this notion, the lumen of the airways was filled with solutions containing various HCO3– concentrations while the bath contained normal 25 mM HCO3– KRB. After 2 h, the HCO3– concentration in the instillates that had initially contained 150 mM HCO3– had fallen to 54.3 ± 6.7 meq/l (n = 6) (Fig. 6). When the instillates were initially HCO3–-free, the HCO3– concentration rose to nearly that of the KRB bath (18.3 ± 2.6 meq/l, n = 6) after 2 h. When the instillates were normal KRB (identical to the bath), the HCO3– concentration in the instillate was 22.1 ± 2.2 meq/l (n = 6). It was thus likely that some magnitude of HCO3– equilibration occurred between the secreted luminal liquid and the KRB bath during the course of the experiments.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONAPPENDIXGRANTSREFERENCES |
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50% by exposure to Cl–-free solution. However, induction of HCO3– secretion by Cl–-free solution, as judged by the liquid secretion response and the increase in HCO3– concentration, must have been much weaker than that seen with bumetanide. We cannot presently account for this discrepancy since both bumetanide and Cl–-free treatments should reduce Na+, K+, and Cl– influx across the basolateral membrane through NKCC. We speculate that the stimulus for both NKCC and NHE activation is cell shrinkage, which likely occurs immediately following stimulation (8). Perhaps pharmacological blockade of NKCC leads to an exaggerated cell shrinkage response that maximizes NHE activity. Extracellular Cl–-free solution does not simply block influx of these ions across the basolateral membrane of these cells; this maneuver should reverse the net driving force so that ion efflux through NKCC is favored, a condition that might somehow minimize or prevent NHE induction. Alternatively, if the anion exchanger AE2 is present in the basolateral membrane as reported for Calu-3 cells and rat tracheal serous cells (18), it is possible that extracellular Cl–-free solution could drive significant influx of HCO3– through this anion exchanger, thus alkalinizing the cytoplasm sufficiently to prevent activation of NHE. It is worthy of note that Joo and coworkers (14), who measured forskolin- and carbachol-induced liquid secretion from individual glands in porcine tracheae, report no evidence of HCO3– secretion induction with either bumetanide or Cl–-free solution. We are currently unable to account for this discrepancy; however, we are confident in our observation since we have seen evidence of bumetanide induction of HCO3– secretion in our prior studies of both acetylcholine- and substance P-induced secretion in porcine bronchi (2, 10, 29, 30). Our results indicate that DIDS most likely reduced the Jv response to forskolin by inhibiting a component of HCO3– secretion. This notion is evidenced by the parallel fall in HCO3– concentration in the secreted liquid and the apparent sensitivity of the DIDS component to HCO3– removal in the presence of bumetanide. It is unclear, however, which ion transporter was targeted by DIDS to induce this effect. DIDS is known to inhibit AE (24). Recycling Cl– across the basolateral membrane through NKCC and AE2 could theoretically drive transepithelial HCO3– secretion through this anion exchanger; however, according to this mechanism, DIDS-sensitive HCO3– secretion should be dependent on NKCC activity and thus inhibited by bumetanide, which does not appear to be the case from our data. Moreover, the stoichiometry of these anion exchangers is 1:1; therefore, no net charge or net osmolyte transfer should be achieved by anion exchange activity alone across either the apical or basolateral membranes. DIDS is also known to inhibit NBC and NDAE transporters (24), which could drive HCO3– entry across the basolateral membrane of secretory cells. Involvement of either of these transporters is more likely since they could account for the extracellular Na+ dependence of this component of the forskolin-induced Jv. In support of our findings, Devor and coworkers (6) observed in Calu-3 cells that forskolin induced HCO3– secretion that appeared to be driven by a basolateral membrane NBC cotransporter in that this process was Na+ dependent and inhibited by a related stilbene inhibitor, 4,4'-dinitrostilbene-2,2'-disulfonic acid. Furthermore, mRNA for NBC1 has been identified in Calu-3 cells (9), and this transporter has been shown to be activated by cAMP in porcine vas deferens (4) as well as murine colonic crypts (1). However, in contrast to our findings from the present study, forskolin alone apparently does not induce bumetanide-sensitive Cl–-secretion in Calu-3 cells (4).
Because of the lack of selectivity of these anion channel blockers, it is not possible to identify the specific anion channel(s) involved by the secretion response to any single inhibitor. However, the responses to a spectrum of inhibitors and agonists can provide important clues as to the channels that most likely participate in the secretion process of glands. In the present study, NPPB, DPC, and glibenclamide significantly reduced the forskolin-induced Jv. These agents have been all been recognized as inhibitors of CFTR though their selectivity among different classes of anion channels is poor (25). Niflumic acid, which is often used as a probe of Ca2+-activated Cl– channels, had no effect on forskolin-induced secretion in the present study. Although niflumic acid is capable of blocking CFTR, it is more potent against Ca2+-activated Cl– channels and is a less potent blocker of CFTR than NPPB (26). CFTR is activated when phosphorylated by protein kinase A (5); consequently, forskolin, which increases cAMP production through direct stimulation of adenylyl cyclase, is often used as a probe of CFTR activity. The endogenous agonist for CFTR-dependent secretion in normal airway glands is probably VIP, since submucosal glands from human CF airways lose the ability to respond to either forskolin or VIP (13). Although our results are not definitive, the aggregate data support a role for CFTR in the forskolin-induced Jv. We cannot discount the possibility that DIDS, which blocks numerous non-CFTR anion channels, reduces a component of the Jv through blockade of a non-CFTR, cAMP-regulated anion channel. But, aside from possible species differences, the existence of a non-CFTR, cAMP-activated channel in the apical membrane of the secretory cells would be surprising since glands from excised CF airways secrete no liquid when exposed to forskolin; therefore, all liquid that is induced by this agonist likely depends upon the presence of functional CFTR.
Because forskolin is a direct activator of adenylyl cyclase, we expected that the secretion response to this agonist would be a purely cAMP-dependent response. However, our observation that the forskolin-induced secretion response is dependent upon bath Ca2+ challenges this notion. The identity of the Ca2+-dependent element in this signal transduction cascade is currently unknown, but several possibilities exist. Populations of apical membrane anion channels, basolateral membrane K+ channels, or intracellular kinases might be directly activated by increases in intracellular Ca2+ concentration, or they may be secondarily activated or regulated by calmodulin. Additional studies will be needed to resolve this issue.
In the present study, HCO3– concentrations in the airway liquid samples were measured as a means for assessing the role of this anion in the liquid secretion process. We found that the airway epithelial barrier could not maintain large concentration gradients for HCO3– over the period of our experimental protocol. Because the directional changes in luminal HCO3– concentrations appeared to mirror the HCO3– concentration gradients imposed across the airway wall, we expect that these changes were due in large in part to partial equilibration of this anion across the surface epithelium of the airways. Indeed, sheep tracheal epithelium is sufficiently permeable to Cl– to permit a similar magnitude of anion efflux (see APPENDIX); thus the HCO3– "leak" that we see in the present study could be explained simply if the airway epithelial barrier were similarly permeable to Cl– and HCO3–. Alternatively, it is possible that a fraction of the loss of HCO3– from the 150 mM HCO3– instillate was due to the secretion of acid. Inglis et al. (10) reported that isolated perfused porcine bronchi secrete acid equivalents by a bafilomycin A1-sensitive process that is induced by luminal alkalinity. Regardless of the mechanism, this finding in the present study accounts for our previous observations with other secretion agonists where HCO3– concentrations in airway liquid were lower than expected following inhibition of Cl– secretion with bumetanide (3, 30). Thus the luminal liquid HCO3– concentrations that are reported here must be recognized to be the consequence of both the primary glandular secretory processes and the secondary surface epithelial processes (i.e., transepithelial equilibration perhaps influenced by acid secretion). Because significant differences in HCO3– concentrations were observed between inhibitor treatments and their paired controls and because the changes trended in predictable directions, we believe that the HCO3– concentrations in airway liquid provide useful interpretive information. However, it is clear that these data should be judged cautiously.
Several similarities were found between the mechanism of forskolin-induced secretion and those previously reported for acetylcholine and substance P in these bronchi. All three secretagogues exhibit NKCC-dependent Cl– secretion, and, in the presence of bumetanide, NHE-dependent HCO3– secretion is observable (3, 29, 30). Furthermore, the Jv response is sensitive to NPPB, DPC, and glibenclamide (3, 29). These results indicate that cAMP-mediated secretion utilizes several components common to acetylcholine- and substance P-dependent secretion. In contrast to our previous studies with acetylcholine and substance P, we noted a significant DIDS-sensitive component in forskolin-induced secretion (3, 29). As discussed above, the target for this agent is unclear, but we speculate that DIDS inhibits a basolateral membrane HCO3– cotransporter such as NBC or NDAE.
In conclusion, we report that forskolin induces liquid secretion from the submucosal glands of porcine bronchial airways through induction of Cl– and HCO3– secretion. We note several important similarities between this process and muscarinic-induced liquid secretion including participation of NKCC and NHE as respective mediators of Cl– and HCO3– secretion. One noticeable difference was a prominent effect of DIDS, which blocks a component of HCO3– secretion. Because forskolin is likely to have induced secretion through elevation of intracellular cAMP, we speculate that CFTR serves as the anion efflux pathway across the apical membrane of the glandular secretory cells.
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APPENDIX |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONAPPENDIXGRANTSREFERENCES |
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C = 125 meq/l) that was created in our 150 meq/l HCO3– instillate experiments. Chloride flux (JCl) is estimated from the following derivation of the Fick equation:
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GRANTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONAPPENDIXGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONAPPENDIXGRANTSREFERENCES |
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F508 cystic fibrosis transmembrane regulator in human respiratory epithelia. Mol Biol Cell 16: 2154–2167, 2005.This article has been cited by other articles:
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  C. S. Rogers, W. M. Abraham, K. A. Brogden, J. F. Engelhardt, J. T. Fisher, P. B. McCray Jr., G. McLennan, D. K. Meyerholz, E. Namati, L. S. Ostedgaard, et al. The porcine lung as a potential model for cystic fibrosis Am J Physiol Lung Cell Mol Physiol, August 1, 2008; 295(2): L240 - L263. [Abstract] [Full Text] [PDF]
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A. S. Verkman From the farm to the lab: the pig as a new model of cystic fibrosis lung disease Am J Physiol Lung Cell Mol Physiol, August 1, 2008; 295(2): L238 - L239. [Full Text] [PDF]
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 J. P. Ianowski, J. Y. Choi, J. J. Wine, and J. W. Hanrahan Mucus secretion by single tracheal submucosal glands from normal and cystic fibrosis transmembrane conductance regulator knockout mice J. Physiol., April 1, 2007; 580(1): 301 - 314. [Abstract] [Full Text] [PDF]
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 X. Liu, M. Luo, L. Zhang, W. Ding, Z. Yan, and J. F. Engelhardt Bioelectric Properties of Chloride Channels in Human, Pig, Ferret, and Mouse Airway Epithelia Am. J. Respir. Cell Mol. Biol., March 1, 2007; 36(3): 313 - 323. [Abstract] [Full Text] [PDF]
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