Hyposecretion, Not Hyperabsorption, Is the Basic Defect of Cystic Fibrosis Airway Glands
2006; Elsevier BV; Volume: 281; Issue: 11 Linguagem: Inglês
10.1074/jbc.m512766200
ISSN1083-351X
AutoresNam Soo Joo, Toshiya Irokawa, Robert C. Robbins, Jeffrey J. Wine,
Tópico(s)Ion Transport and Channel Regulation
ResumoHuman airways and glands express the anion channel cystic fibrosis transmembrane conductance regulator, CFTR, and the epithelial Na+ channel, ENaC. Cystic fibrosis (CF) airway glands fail to secrete mucus in response to vasoactive intestinal peptide or forskolin; the failure was attributed to loss of CFTR-mediated anion and fluid secretion. Alternatively, CF glands might secrete acinar fluid via CFTR-independent pathways, but the exit of mucus from the glands could be blocked by hyperabsorption of fluid in the gland ducts. This could occur because CFTR loss can disinhibit ENaC, and ENaC activity can drive absorption. To test these two hypotheses, we measured single gland mucus secretion optically and applied ENaC inhibitors to determine whether they augmented secretion. Human CF glands were pretreated with benzamil and then stimulated with forskolin in the continued presence of benzamil. Benzamil did not rescue the lack of secretion to forskolin (50 glands, 6 CF subjects) nor did it increase the rate of cholinergically mediated mucus secretion from CF glands. Finally, neither benzamil nor amiloride increased forskolin-stimulated mucus secretion from porcine submucosal glands (75 glands, 7 pigs). One possible explanation for these results is that ENaC within the gland ducts was not active in our experiments. Consistent with that possibility, we discovered that human airway glands express Kunitz-type and non-Kunitz serine protease inhibitors, which might prevent proteolytic activation of ENaC. Our results suggest that CF glands do not display excessive, ENaC-mediated fluid absorption, leaving defective, anion-mediated fluid secretion as the most likely mechanism for defective mucus secretion from CF glands. Human airways and glands express the anion channel cystic fibrosis transmembrane conductance regulator, CFTR, and the epithelial Na+ channel, ENaC. Cystic fibrosis (CF) airway glands fail to secrete mucus in response to vasoactive intestinal peptide or forskolin; the failure was attributed to loss of CFTR-mediated anion and fluid secretion. Alternatively, CF glands might secrete acinar fluid via CFTR-independent pathways, but the exit of mucus from the glands could be blocked by hyperabsorption of fluid in the gland ducts. This could occur because CFTR loss can disinhibit ENaC, and ENaC activity can drive absorption. To test these two hypotheses, we measured single gland mucus secretion optically and applied ENaC inhibitors to determine whether they augmented secretion. Human CF glands were pretreated with benzamil and then stimulated with forskolin in the continued presence of benzamil. Benzamil did not rescue the lack of secretion to forskolin (50 glands, 6 CF subjects) nor did it increase the rate of cholinergically mediated mucus secretion from CF glands. Finally, neither benzamil nor amiloride increased forskolin-stimulated mucus secretion from porcine submucosal glands (75 glands, 7 pigs). One possible explanation for these results is that ENaC within the gland ducts was not active in our experiments. Consistent with that possibility, we discovered that human airway glands express Kunitz-type and non-Kunitz serine protease inhibitors, which might prevent proteolytic activation of ENaC. Our results suggest that CF glands do not display excessive, ENaC-mediated fluid absorption, leaving defective, anion-mediated fluid secretion as the most likely mechanism for defective mucus secretion from CF glands. Mucus clearance is an important component of airway innate defenses, and defects in clearance appear to be an important problem in cystic fibrosis (CF) 2The abbreviations used are: CF, cystic fibrosis; CFTR, CF transmembrane conductance regulator; ENaC, epithelial Na+ channel; VIP, vasoactive intestinal peptide; 1-EBIO, 1-ethyl-benzimidazolinone; KRB, Krebs-Ringer bicarbonate; HAI, hepatocyte growth factor activator inhibitor; NEI, inhibitor of human neutrophil elastase.2The abbreviations used are: CF, cystic fibrosis; CFTR, CF transmembrane conductance regulator; ENaC, epithelial Na+ channel; VIP, vasoactive intestinal peptide; 1-EBIO, 1-ethyl-benzimidazolinone; KRB, Krebs-Ringer bicarbonate; HAI, hepatocyte growth factor activator inhibitor; NEI, inhibitor of human neutrophil elastase. airway disease (1Knowles M.R. Boucher R.C. J. Clin. Invest. 2002; 109: 571-577Crossref PubMed Scopus (949) Google Scholar). Submucosal glands are the major sites for mucus production in large airways. They secrete the gel-forming mucin MUC5B, many innate defense molecules, and the serine protease inhibitors α-1-antitrypsin and α-1-antichymotrypsin (2Joo N.S. Lee D.J. Winges K.M. Rustagi A. Wine J.J. J. Biol. Chem. 2004; 279: 38854-38860Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The innate mucosal defense system, which includes mucociliary and cough clearance, effectively entraps, inactivates, and removes airborne pathogens to maintain sterile airways (1Knowles M.R. Boucher R.C. J. Clin. Invest. 2002; 109: 571-577Crossref PubMed Scopus (949) Google Scholar, 3Wine J.J. Joo N.S. Proc. Am. Thorac. Soc. 2004; 1: 47-53Crossref PubMed Scopus (184) Google Scholar). These vital host defenses are compromised in CF airways, allowing pathogens to grow within static airway mucus eventually destroying the lungs. Human CF airway glands fail to secrete mucus in response to vasoactive intestinal peptide VIP, or forskolin (4Joo N.S. Irokawa T. Wu J.V. Robbins R.C. Whyte R.I. Wine J.J. J. Biol. Chem. 2002; 277: 50710-50715Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), but continue to secrete in response to cholinergic stimulation. However, the properties of CF gland mucus produced in response to carbachol are altered (5Jayaraman S. Joo N.S. Reitz B. Wine J.J. Verkman A.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8119-8123Crossref PubMed Scopus (162) Google Scholar), and their responses appear to be reduced when corrected for the hypertrophied state of CF glands. 3N. S. Joo and J. J. Wine, unpublished results.3N. S. Joo and J. J. Wine, unpublished results. These results have been independently confirmed (6Salinas D. Haggie P.M. Thiagarajah J.R. Song Y. Rosbe K. Finkbeiner W.E. Nielson D.W. Verkman A.S. FASEB J. 2005; 19: 431-433Crossref PubMed Scopus (66) Google Scholar, 7Song Y. Salinas D. Nielson D.W. Verkman A.S. Am. J. Physiol. 2006; (in press)Google Scholar). The direct explanation for these results is that CFTR-mediated fluid secretion is lost in CF glands. However, both CFTR and ENaC are expressed in human submucosal glands (8Burch L.H. Talbot C.R. Knowles M.R. Canessa C.M. Rossier B.C. Boucher R.C. Am. J. Physiol. 1995; 269: C511-C518Crossref PubMed Google Scholar, 9Engelhardt J.F. Yankaskas J.R. Ernst S.A. Yang Y. Marino C.R. Boucher R.C. Cohn J.A. Wilson J.M. Nat. Genet. 1992; 2: 240-248Crossref PubMed Scopus (567) Google Scholar, 10Gaillard D. Hinnrasky J. Coscoy S. Hofman P. Matthay M.A. Puchelle E. Barbry P. Am. J. Physiol. 2000; 278: L177-L184Crossref PubMed Google Scholar, 11Kreda S.M. Mall M. Mengos A. Rochelle L. Yankaskas J. Riordan J.R. Boucher R.C. Mol. Biol. Cell. 2005; 16: 2154-2167Crossref PubMed Scopus (220) Google Scholar), and primary cultures of human large airway submucosal gland acini showed evidence of both CFTR-dependent Cl- transport and amiloride-sensitive Na+ transport (12Yamaya M. Finkbeiner W.E. Widdicombe J.H. Am. J. Physiol. 1991; 261: L485-L490PubMed Google Scholar).What role does ENaC play in submucosal glands, and how is it regulated? Nothing is known about the activity or function of ENaC within submucosal glands, but in the ciliated cells of the airway surface ENaC is complexly regulated. For example, CFTR expression appears to inhibit ENaC in the airways via an unknown mechanism, while ENaC is activated extracellularly by diverse membrane proteases (13Vallet V. Chraibi A. Gaeggeler H.P. Horisberger J.D. Rossier B.C. Nature. 1997; 389: 607-610Crossref PubMed Scopus (450) Google Scholar, 14Caldwell R.A. Boucher R.C. Stutts M.J. Am. J. Physiol. 2004; 286: C190-C194Crossref PubMed Scopus (169) Google Scholar, 15Bridges R.J. Newton B.B. Pilewski J.M. Devor D.C. Poll C.T. Hall R.L. Am. J. Physiol. 2001; 281: L16-L23Crossref PubMed Google Scholar) and is deactivated by protease inhibitors (13Vallet V. Chraibi A. Gaeggeler H.P. Horisberger J.D. Rossier B.C. Nature. 1997; 389: 607-610Crossref PubMed Scopus (450) Google Scholar, 14Caldwell R.A. Boucher R.C. Stutts M.J. Am. J. Physiol. 2004; 286: C190-C194Crossref PubMed Scopus (169) Google Scholar, 15Bridges R.J. Newton B.B. Pilewski J.M. Devor D.C. Poll C.T. Hall R.L. Am. J. Physiol. 2001; 281: L16-L23Crossref PubMed Google Scholar, 16Caldwell R.A. Boucher R.C. Stutts M.J. Am. J. Physiol. 2005; 288: L813-L819Crossref PubMed Scopus (210) Google Scholar). The function of ENaC within glands is of increased interest because of a recent study which suggested that gland acini contain only scant CFTR (11Kreda S.M. Mall M. Mengos A. Rochelle L. Yankaskas J. Riordan J.R. Boucher R.C. Mol. Biol. Cell. 2005; 16: 2154-2167Crossref PubMed Scopus (220) Google Scholar), while the ducts contain abundant CFTR, raising the question of how the non-responsiveness of CF glands can be explained. One possibility is that the ducts actually secrete the glandular fluid. Another is that the co-localization of ENaC and CFTR in the ciliated ducts of glands may mean that ENaC is disinhibited in CF glands by the loss of CFTR (17Stutts M.J. Canessa C.M. Olsen J.C. Hamrick M. Cohn J.A. Rossier B.C. Boucher R.C. Science. 1995; 269: 847-850Crossref PubMed Scopus (952) Google Scholar), leading to increased volume absorption of fluid from CF glands. That might be sufficient to block secretion from CF glands.To test this latter hypothesis, we used reverse transcriptase PCR to confirm the presence of ENaC subunits in human submucosal glands and then used optical methods to study secretion rates of single submucosal glands from CF subjects and from normal pigs in the presence or absence of ENaC inhibitors. We found no evidence that ENaC inhibitors altered secretion rates in either normal or CF glands. To try to understand why this might be, we used reverse transcriptase PCR to show that glands express multiple serine protease inhibitors. These results are discussed in terms of a hypothesis that innate anti-serine proteases modify ENaC activity both within glands and on the surface of the airways.EXPERIMENTAL PROCEDURESAirway Tissues and Cell Cultures—Human CF lungs and tracheal scraps from donors were obtained from lung transplants or, in one CF case, from an autopsy specimen harvested less than 2 h post mortem at Stanford Hospital. The average ages of the CF transplants were 29 ± 2 years, and three of them were male. Our study was approved by the Institutional Review Board of Stanford University. Post-mortem (<1 h) Yorkshire pig tracheas were obtained from the animal facility at Stanford University after acute experiments unrelated to our studies. The human airway gland serous cell line, Calu-3, was maintained as described previously (2Joo N.S. Lee D.J. Winges K.M. Rustagi A. Wine J.J. J. Biol. Chem. 2004; 279: 38854-38860Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The human airway surface epithelial cell line, H441, was kindly provided by Rabin Tirouvanziam, Stanford University, and maintained in a T25 tissue culture flask containing a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture supplemented with 10% fetal bovine serum (Sigma), 100 μg/ml streptomycin, 100 units/ml penicillin, and 2 mm glutamine at 37 °C in a humidified atmosphere containing 5% CO2. Cells were passaged once at 7-10 days with a density of ∼1.5 × 104/cm2.Tissue Preparations and Optical Measurements—Harvested tissues were kept until use in ice-cold Krebs-Ringer bicarbonate (KRB) buffer gassed with 95% O2 and 5% CO2. The KRB buffer composition was 115 mm NaCl, 2.4 mm K2HPO4, 0.4 mm KH2PO4, 25 mm NaHCO3, 1.2 mm MgCl2, 1.2 mm CaCl2, and 10 mm glucose (pH 7.4) adjusted to ∼290 mosm with a Wescor vapor pressure osmometer. To minimize endogenously generated prostaglandins during tissue preparation, 1.0 μm indomethacin was present in the Krebs buffer. Tissue preparation for optical measurements and the experimental setup for single gland mucus secretion measurements were described previously (4Joo N.S. Irokawa T. Wu J.V. Robbins R.C. Whyte R.I. Wine J.J. J. Biol. Chem. 2002; 277: 50710-50715Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 18Joo N.S. Saenz Y. Krouse M.E. Wine J.J. J. Biol. Chem. 2002; 277: 28167-28175Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 19Joo N.S. Wu J.V. Krouse M.E. Saenz Y. Wine J.J. Am. J. Physiol. 2001; 281: L458-L468Crossref PubMed Google Scholar). Briefly, a piece of dissected human CF bronchial preparation was mounted as apical-side-up in a Sylgard-lined 35-mm plastic Petri dish and placed onto an optical chamber where temperature and pH are controlled. The surface of the tissue was cleaned, dried, and covered with water-saturated mineral oil. The rate at which spherical mucus bubbles were secreted from the gland ducts into the oil layer was optically recorded at intervals of 1-5 min using a computer-controlled Nikon digital camera. Stored images were analyzed by Scion Image software (Scion Corp.). To determine the effects of ENaC blockers, tissue preparations were pretreated with 10 μm amiloride or 10 μm benzamil for various durations (5-60 min) and exposed either apically only or apically + basolaterally. They were then stimulated with 10 μm forskolin in the continued presence of the inhibitor in the bath.Reverse Transcriptase PCR—Total RNA was extracted from isolated human airway glands, Calu-3 cells, and H441 cells using the Qiagen total RNA isolation kit (Qiagen, CA). Primer pairs were designed to span an intron whenever possible as an additional precaution against contamination by genomic templates. The primer sequences, expected sizes (bp) of the amplicons, and GenBank™ reference sequence accession numbers used for designing primers are shown in Table 1. The PCR amplifications were done for 35 cycles of 45 s at 94 °C followed by 45 s at 53-56 °C and 60 s at 72 °C using the Qiagen HotStarTaq DNA polymerase kit (Qiagen, CA). PCR products were separated on a 1.5% agarose gel and then visualized by ethidium bromide staining.TABLE 1PCR primer sequences of ENaC and serine protease inhibitors RefSeq represents a reference sequence of GenBank™ accession number used for designing primers or cited reference in this study. HAI, TFPI, IaTI, and NEI are the abbreviations for: hepatocyte growth factor activator inhibitor, tissue factor pathway inhibitor, inter-α -trypsin inhibitor, and inhibitor of neutrophil elastase, respectively.Primer setSequenceProduct sizeRefSeqbpENaC-α forward5′-CCTGGAATCAACAACGGTCT-3′188NM 001038ENaC-α reverse5′-AGGGTTTCCTTCCTCATGCT-3′ENaC-β forward5′-GACCATCTCCATGGCTGACT-3′173NM 000336ENaC-β reverse5′-TGGCTGCTGATTCTTCAATG-3′ENaC-γ forward5′-CACCATCTGCAACATCAACC-3′155NM 001039ENaC-γ reverse5′-CCTCTGAGACGGAGTTCCAG-3′HAI-1 forward5′-ATGGAGGCTGCTTGGGCAACA-3′221Ref. 29Kirchhofer D. Peek M. Li W. Stamos J. Eigenbrot C. Kadkhodayan S. Elliott J.M. Corpuz R.T. Lazarus R.A. Moran P. J. Biol. Chem. 2003; 278: 36341-36349Abstract Full Text Full Text PDF PubMed Scopus (85) Google ScholarHAI-1 reverse5′-ACAGGCAGCCTCGTCGGAGG-3′HAI-1B forward5′-ATGGAGGCTGCTTGGGCAACA-3′269Ref. 29Kirchhofer D. Peek M. Li W. Stamos J. Eigenbrot C. Kadkhodayan S. Elliott J.M. Corpuz R.T. Lazarus R.A. Moran P. J. Biol. Chem. 2003; 278: 36341-36349Abstract Full Text Full Text PDF PubMed Scopus (85) Google ScholarHAI-1B reverse5′-ACAGGCAGCCTCGTCGGAGG-3′HAI-2 forward5′-AACAGCTACCGCTCTGAGGA-3′233NM 021102HAI-2 reverse5′-TTCTTCACCAGCTGCTCCTT-3′TFPI forward5′-AGATACGGAGTTGCCACCAC-3′150NM 006287TFPI reverse5′-TTTCCTTCACATCCCCCATA-3′TFPI-2 forward5′-GTCGATTCTGCTGCTTTTCC-3′441NM006528TFPI-2 reverse5′-ATGGAATTTTCTTTGGTGCG-3′IαTI forward5′-CAGGACGTTTGTGAAGAGCA-3′244NM 002217IαTI reverse5′-GCCAAAGCCCAGGTTATACA-3′SerpinF2 forward5′-GCAGAAAGGATTTCCCATCA-3′237NM 000934SerpinF2 reverse5′-GGGTCAAACTTGTTCCTCCA-3′NEI forward5′-TTCAAGGGAAACTGGAAGGA-3′223NM 030666NEI reverse5′-TGGACTCGTCCTCAATGTCA-3′ Open table in a new tab Electrophysiology—Porcine tracheal tissue preparations were mounted in EasyMount Ussing chambers (Physiologic Instruments, San Diego, CA) with an exposed surface of 0.45 cm2 and bathed in KRB buffer. Transepithelial short circuit current (Isc) was measured using a VCC-600 voltage clamp (Physiologic Instruments), and Isc data were captured and displayed with PowerLab Chart4 software (AD Instruments, Mountain View, CA). Tissue preparations were pretreated with 10 μm benzamil or a vehicle before being mounted in the chambers and variously stimulated or inhibited with 0.1 mm carbachol, 1 mm 1-ethyl-benzimidazolinone (1-EBIO), 0.1 mm bumetanide, and 20 μm ouabain to an apical or basolateral chamber as indicated in the legend to Fig. 2.Reagents—PCR kits were stored according to manufacturer's protocols. Chemicals (Sigma) were made fresh or maintained at -20 °C as aliquots of stock concentrations. Stock solutions of carbachol, amiloride, ouabain, and VIP were made in deionized water, while forskolin, benzamil, and 1-EBIO were dissolved in dimethyl sulfoxide (Me2SO).Statistics—Data are expressed as mean ± S.E. unless indicated otherwise. Student's t test for unpaired data was used to compare means of different treatment groups. The difference between the two means was considered to be significant when p < 0.05.RESULTS AND DISCUSSIONConfirmation of Minimal Responses of CF Glands Stimulated with VIP/Forskolin—Human airway glands from donors and from patients with lung diseases other than CF, as well as airway glands from pigs, secret mucus in response to VIP or forskolin (4Joo N.S. Irokawa T. Wu J.V. Robbins R.C. Whyte R.I. Wine J.J. J. Biol. Chem. 2002; 277: 50710-50715Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 18Joo N.S. Saenz Y. Krouse M.E. Wine J.J. J. Biol. Chem. 2002; 277: 28167-28175Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 20Ballard S.T. Inglis S.K. J. Physiol. (Lond.). 2004; 556: 1-10Crossref Scopus (82) Google Scholar). In marked contrast, glands from CF subjects show virtually no response to these agents, even though they continue to respond to carbachol (4Joo N.S. Irokawa T. Wu J.V. Robbins R.C. Whyte R.I. Wine J.J. J. Biol. Chem. 2002; 277: 50710-50715Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The lack of mucus secretion in response to VIP and forskolin shows in Fig. 1A, while carbachol produced a good response (sustained carbachol secretion rates: 5.38 ± 1.08 nl/min for 8 glands).FIGURE 1Lack of mucus secretion in response to cAMP agonists in CF airways. A, the CF gland preparation was initially stimulated 2 μm carbachol to clear out potentially clogged CF gland ducts followed by thorough washout of carbachol from bath solution. The glands then were stimulated with 1 μm VIP and 10 μm forskolin (Fsk) for 60 min and were subsequently stimulated with 10 μm carbachol (Carb). The arrows show the time points when the corresponding drugs are added. While no increase is found with VIP + forskolin, the carbachol treatment causes mucus secretion. A representative time course is shown here in an analyzed subset of 151 glands from 18 different CF subjects. B, two isolated human airway submucosal glands from the tracheal preparation of healthy donors. Both glands were stained with Neutral Red dye to visualize them, and the one on the right clearly shows ductal parts of a gland after being injected with Brilliant Blue dye into its ductal opening. Scale is 0.5 mm. C, reverse transcriptase PCR of total RNA from isolated human submucosal glands (Hm-SMG), Calu-3 human gland serous cell lines, and H441 human airway surface cells was carried out using primer sets that encompass at least one intron. The expected amplicon sizes of α-, β-, and γ-ENaC subunits are 188, 173, and 155 bp, respectively. All three subunits exist in human submucosal glands and H441 cells, and only a-ENaC subunit is found in Calu-3 cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In an attempt to detect any small responses to VIP or forskolin that might be present in CF glands, and to distinguish between responses and the unstimulated or "basal" secretion often observed in glands, we used our previously developed optical methods (4Joo N.S. Irokawa T. Wu J.V. Robbins R.C. Whyte R.I. Wine J.J. J. Biol. Chem. 2002; 277: 50710-50715Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 18Joo N.S. Saenz Y. Krouse M.E. Wine J.J. J. Biol. Chem. 2002; 277: 28167-28175Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 19Joo N.S. Wu J.V. Krouse M.E. Saenz Y. Wine J.J. Am. J. Physiol. 2001; 281: L458-L468Crossref PubMed Google Scholar) to analyze single gland mucus secretion rates in response to forskolin/VIP from a subset of 151 glands among more than 1000 observed glands from 18 different CF subjects. These glands were selected based on the presence of mucus bubbles prior to carbachol stimulation, which ensured that the ducts were not blocked. The mean secretion rate in response to forskolin/VIP in this set of CF glands was 0.01 ± 0.01 nl/min. This contrasts with response rates to forskolin of 0.94 ± 0.19 nl/min for 139 glands from 16 normal donor control subjects and 0.99 ± 0.23 nl/min for 79 glands from 12 disease control subjects. This ∼100-fold difference in response rates was previously interpreted to be the result of the loss of CFTR in CF gland serous cells and the consequent absence of the HCO3- and Cl--mediated fluid secretion thought to occur via CFTR (4Joo N.S. Irokawa T. Wu J.V. Robbins R.C. Whyte R.I. Wine J.J. J. Biol. Chem. 2002; 277: 50710-50715Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). This interpretation is based on the assumption that Calu-3 cells, which secrete copious fluid in response to VIP and forskolin (21Irokawa T. Krouse M.E. Joo N.S. Wu J.V. Wine J.J. Am. J. Physiol. 2004; 287: L784-L793Crossref PubMed Scopus (23) Google Scholar), are a reasonable model for gland serous cells, as well as on the observation that gland serous cells express abundant CFTR (9Engelhardt J.F. Yankaskas J.R. Ernst S.A. Yang Y. Marino C.R. Boucher R.C. Cohn J.A. Wilson J.M. Nat. Genet. 1992; 2: 240-248Crossref PubMed Scopus (567) Google Scholar).However, a reinvestigation of human airways with a new set of monoclonal antibodies found only sparse and variable CFTR in the gland serous cells but abundant CFTR in the airway surface and in the ducts (11Kreda S.M. Mall M. Mengos A. Rochelle L. Yankaskas J. Riordan J.R. Boucher R.C. Mol. Biol. Cell. 2005; 16: 2154-2167Crossref PubMed Scopus (220) Google Scholar). The different, antibody-dependent distributions of CFTR remain to be resolved. One approach to resolving the differences is to use physiology to assess some possible functional consequences of each pattern. Because CFTR in the ducts is accompanied by ENaC (10Gaillard D. Hinnrasky J. Coscoy S. Hofman P. Matthay M.A. Puchelle E. Barbry P. Am. J. Physiol. 2000; 278: L177-L184Crossref PubMed Google Scholar, 11Kreda S.M. Mall M. Mengos A. Rochelle L. Yankaskas J. Riordan J.R. Boucher R.C. Mol. Biol. Cell. 2005; 16: 2154-2167Crossref PubMed Scopus (220) Google Scholar), and because of evidence that the loss of CFTR increases ENaC activity in CF airways (17Stutts M.J. Canessa C.M. Olsen J.C. Hamrick M. Cohn J.A. Rossier B.C. Boucher R.C. Science. 1995; 269: 847-850Crossref PubMed Scopus (952) Google Scholar), an alternative explanation for the lack of CF gland secretion is that VIP/forskolin still stimulates CF glands to secrete fluid via non-CFTR pathways, but the lack of observed fluid secretion from the openings of CF gland ducts results from enhanced, ENaC-driven fluid volume absorption in the CF gland ducts. The volume absorption need not completely offset secretion; it could be that hyperabsorption in the small diameter ducts could lead to blockage.Confirmation of ENaC Expression in Human Airway Glands—Two prior studies, taken together, provided evidence for ENaC expression in glands, but neither study alone demonstrated expression of α-, β-, and γ-ENaC subunits (8Burch L.H. Talbot C.R. Knowles M.R. Canessa C.M. Rossier B.C. Boucher R.C. Am. J. Physiol. 1995; 269: C511-C518Crossref PubMed Google Scholar, 10Gaillard D. Hinnrasky J. Coscoy S. Hofman P. Matthay M.A. Puchelle E. Barbry P. Am. J. Physiol. 2000; 278: L177-L184Crossref PubMed Google Scholar). Fig. 1B shows two representative isolated human submucosal glands after injections with Brilliant Blue (right one: note dark blue collecting ductal parts) followed by whole gland staining with Neutral Red dye to more clearly visualize the glands and permit certainty that the tissue was glandular. Reverse transcriptase PCR for α-, β-, and γ-ENaC subunits were carried out using human airway submucosal glands, Calu-3 cells, and H441 human airway surface epithelial cells as sources for RNA. We extracted total RNA from each sample and ran reverse transcriptase PCR as described. All three ENaC subunits were demonstrated in RNA from isolated human glands and from H441 surface epithelial cells (Fig. 1C). Calu-3 cells only expressed the α-ENaC subunit. Calu-3 cells lack amiloride-sensitive Na+ transport (22Singh M. Krouse M. Moon S. Wine J.J. Am. J. Physiol. 1997; 272: L690-L698PubMed Google Scholar). These PCR results confirm previous reports that ENaC is expressed in human airway surface epithelia and submucosal glands (8Burch L.H. Talbot C.R. Knowles M.R. Canessa C.M. Rossier B.C. Boucher R.C. Am. J. Physiol. 1995; 269: C511-C518Crossref PubMed Google Scholar, 10Gaillard D. Hinnrasky J. Coscoy S. Hofman P. Matthay M.A. Puchelle E. Barbry P. Am. J. Physiol. 2000; 278: L177-L184Crossref PubMed Google Scholar).Continued Lack of Responses to Forskolin in CF Glands Exposed to ENaC Inhibitors—To test the hypothesis that defective gland secretion is caused by enhanced ENaC-driven fluid absorption within CF glands, CF airway glands were incubated with a 10 μm concentration of the potent ENaC inhibitor benzamil for 5-60 min and were then stimulated with forskolin in the continued presence of the inhibitor in the bath. If 1) forskolin-stimulated CF glands still secrete fluid via non-CFTR pathways, and if 2) ENaC is active or hyperactive in CF glands as it appears to be in surface epithelia of CF airways, and if 3) ENaC mediates the absorption of fluid volume as it appears to do for surface epithelia (17Stutts M.J. Canessa C.M. Olsen J.C. Hamrick M. Cohn J.A. Rossier B.C. Boucher R.C. Science. 1995; 269: 847-850Crossref PubMed Scopus (952) Google Scholar), then it might be expected that the inhibition of ENaC could rescue, at least partially, forskolin-stimulated fluid secretion of CF glands.Results from two representative experiments with benzamil are shown in Fig. 2. In the first experiment, benzamil was added to a CF tissue containing glands that were secreting low levels of mucus basally. Benzamil treatment did not affect the basal mucus secretion nor was their any significant stimulation of secretion by subsequent treatment with 10 μm forskolin in the presence of benzamil (Fig. 2A). This result is significant because the low rate of basal secretion proves that the ducts were not blocked; hence even small increases in the net rate of fluid flow from the ducts should have been observed if secretion were the net result of opposing volume secretion and ENaC-based volume absorption. However, a potential criticism of this kind of experiment is that the benzamil might not gain access to ENaC in the lumen of the duct.To partially address that concern, some CF gland preparations were pretreated with benzamil for up to 60 min both apically and basolaterally before the preparation was stimulated with forskolin. Despite this prolonged exposure, there was still no rescue of forskolin-stimulated secretion, as shown for a representative preparation treated for 60 min (Fig. 2B). Similar experiments were carried out with a total of 50 glands from 6 different CF subjects with the same results: there was no indication of any increase in either basal secretion, when present, and no rescue of the lack of forskolin-stimulated secretion.To verify tissue viability, the tissue was stimulated with 10 μm carbachol in the presence of benzamil at the end of experiment. The sustained response to carbachol from 5 amiloride or benzamil pretreated subjects was 2.0 ± 0.35 nl/min (40 glands from 5 CF subjects) versus 2.96 ± 0.52 nl/min for carbachol responses from a larger series of CF tissues that were not treated with ENaC inhibitors (143 glands from 17 CF subjects, t test, p = 0.14, not significant).No Effect of Benzamilon Forskolin-stimulated Mucus Secretion from Healthy Pig Glands—To determine whether ENaC-driven volume absorption plays a role in normal airway glands, experiments like those above were also carried out with porcine tracheal preparations. These were pretreated with the ENaC inhibitors benzamil or amiloride with vehicle controls and were then stimulated with 10 μm forskolin in the continued presence or absence of the inhibitors (Fig. 2C). No significant differences in mucus secretion rates were observed in the presence of either ENaC inhibitor. Secretion rates from individual glands were 0.73 ± 0.14 nl/min in the benzamil/amiloride-pretreated group (n = 8, 75 glands from 7 pigs) and 0.67 ± 0.14 nl/min in the control group (n = 8, 75 gland
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