Measurement of Fluid Secretion from Intact Airway Submucosal Glands

  • Jeffrey J. WineEmail author
  • Nam Soo Joo
  • Jae Young Choi
  • Hyung-Ju Cho
  • Mauri E. Krouse
  • Jin V. Wu
  • Monal Khansaheb
  • Toshiya Irokawa
  • Juan Ianowski
  • John W. Hanrahan
  • Alan W. Cuthbert
  • Kim V. Tran
Part of the Methods in Molecular Biology book series (MIMB, volume 742)


Human airways are kept sterile by a mucosal innate defense system that includes mucus secretion. Mucus is secreted in healthy upper airways primarily by submucosal glands and consists of defense molecules mixed with mucins, electrolytes, and water and is also a major component of sputum. Mucus traps pathogens and mechanically removes them via mucociliary clearance while inhibiting their growth via molecular (e.g., lysozyme) and cellular (e.g., neutrophils, macrophages) defenses. Fluid secretion rates of single glands in response to various mediators can be measured by trapping the primary gland mucus secretions in an oil layer, where they form spherical bubbles that can be optically measured at any desired interval to provide detailed temporal analysis of secretion rates. The composition and properties of the mucus (e.g., solids, viscosity, pH) can also be determined. These methods have now been applied to mice, ferrets, cats, pigs, sheep, and humans, with a main goal of comparing gland secretion in control and CFTR-deficient humans and animals.

Key words

Submucosal gland exocrine secretion CFTR mucosal innate defense ion channel ion transporter calcium-activated chloride channel (CaCC) cAMP Ca2+ myoepithelial cell serous cell mucous cell 



We are grateful to the transplant patients and their families whose cooperation provided the tissues needed for studies of human gland secretion. For help in obtaining informed consent from patients we thank D. Weill, N.R. Henig, J. Theodore, T.E. Robinson, M. Wine, and K. Tran. For access to surgical tissues we thank B.A. Reitz, G.J. Berry, R.C. Robbins, R.I. Whyte, and the staff of the Stanford Transplant team. Jennifer Lyons provided useful discussions and comments. Technical help and data analysis were provided by Tracy Hsu, Christina Tseng, Molly Pam, Wei Chen, Sidney Chang, Kim Tran, and Jonathan Chen. The work was supported by NIH Grant DK-51817, the Cystic Fibrosis Foundation, and CFRI.


  1. 1.
    Joo, N. S., Wu, J. V., Krouse, M. E., Saenz, Y., and Wine, J. J. (2001) Optical method for quantifying rates of mucus secretion from single submucosal glands. Am J Physiol Lung Cell Mol Physiol 281, L458–L468.PubMedGoogle Scholar
  2. 2.
    Quinton, P. M. (1979) Composition and control of secretions from tracheal bronchial submucosal glands. Nature 279, 551–552.PubMedCrossRefGoogle Scholar
  3. 3.
    Kreda, S. M., Mall, M., Mengos, A., Rochelle, L., Yankaskas, J., Riordan, J. R., et al. (2005) Characterization of wild-type and deltaF508 cystic fibrosis transmembrane regulator in human respiratory epithelia. Mol Biol Cell 16, 2154–2167.PubMedCrossRefGoogle Scholar
  4. 4.
    Engelhardt, J. F., Yankaskas, J. R., Ernst, S. A., Yang, Y., Marino, C. R., Boucher, R. C., et al. (1992) Submucosal glands are the predominant site of CFTR expression in the human bronchus. Nat Genet 2, 240–248.PubMedCrossRefGoogle Scholar
  5. 5.
    Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., et al. (2006) CFTR regulates phagosome acidification in macrophages and alters bactericidal activity. Nat Cell Biol 8, 933–944.PubMedCrossRefGoogle Scholar
  6. 6.
    Knowles, M. R., and Boucher, R. C. (2002) Mucus clearance as a primary innate defense mechanism for mammalian airways. J Clin Invest 109, 571–577.PubMedGoogle Scholar
  7. 7.
    Bennett, W. D., Olivier, K. N., Zeman, K. L., Hohneker, K. W., Boucher, R. C., and Knowles, M. R. (1996) Effect of uridine 5'-triphosphate plus amiloride on mucociliary clearance in adult cystic fibrosis. Am J Respir Crit Care Med 153, 1796–1801.PubMedGoogle Scholar
  8. 8.
    Rosenfeld, M., Emerson, J., Accurso, F., Armstrong, D., Castile, R., Grimwood, K., et al. (1999) Diagnostic accuracy of oropharyngeal cultures in infants and young children with cystic fibrosis. Pediatr Pulmonol 28, 321–328.PubMedCrossRefGoogle Scholar
  9. 9.
    Roby, B. B., McNamara, J., Finkelstein, M., and Sidman, J. (2008) Sinus surgery in cystic fibrosis patients: comparison of sinus and lower airway cultures. Int J Pediatr Otorhinolaryngol 72, 1365–1369.PubMedCrossRefGoogle Scholar
  10. 10.
    Reid, L. (1960) Measurement of the bronchial mucous gland layer: a diagnostic yardstick in chronic bronchitis. Thorax 15, 132–141.PubMedCrossRefGoogle Scholar
  11. 11.
    Haws, C., Finkbeiner, W. E., Widdicombe, J. H., and Wine, J. J. (1994) CFTR in Calu-3 human airway cells: channel properties and role in cAMP-activated Cl- conductance. Am J Physiol 266, L502–L512.PubMedGoogle Scholar
  12. 12.
    Inglis, S. K., Corboz, M. R., Taylor, A. E., and Ballard, S. T. (1996) Regulation of ion transport across porcine distal bronchi. Am J Physiol 270, L289–L297.PubMedGoogle Scholar
  13. 13.
    Inglis, S. K., Corboz, M. R., Taylor, A. E., and Ballard, S. T. (1997) Effect of anion transport inhibition on mucus secretion by airway submucosal glands. Am J Physiol 272, L372–L377.PubMedGoogle Scholar
  14. 14.
    Inglis, S. K., Corboz, M. R., and Ballard, S. T. (1998) Effect of anion secretion inhibitors on mucin content of airway submucosal gland ducts. Am J Physiol 274, L762–L766.PubMedGoogle Scholar
  15. 15.
    Trout, L., Gatzy, J. T., and Ballard, S. T. (1998) Acetylcholine-induced liquid secretion by bronchial epithelium: role of Cl- and HCO3- transport. Am J Physiol 275, L1095–L1099.PubMedGoogle Scholar
  16. 16.
    Trout, L., King, M., Feng, W., Inglis, S. K., and Ballard, S. T. (1998) Inhibition of airway liquid secretion and its effect on the physical properties of airway mucus. Am J Physiol 274, L258–L263.PubMedGoogle Scholar
  17. 17.
    Ballard, S. T., Trout, L., Bebok, Z., Sorscher, E. J., and Crews, A. (1999) CFTR involvement in chloride, bicarbonate, and liquid secretion by airway submucosal glands. Am J Physiol 277, L694–L699.PubMedGoogle Scholar
  18. 18.
    Trout, L., Corboz, M. R., and Ballard, S. T. (2001) Mechanism of substance P-induced liquid secretion across bronchial epithelium. Am J Physiol Lung Cell Mol Physiol 281, L639–L645.PubMedGoogle Scholar
  19. 19.
    Trout, L., Townsley, M. I., Bowden, A. L., and Ballard, S. T. (2003) Disruptive effects of anion secretion inhibitors on airway mucus morphology in isolated perfused pig lung. J Physiol 549, 845–853.PubMedCrossRefGoogle Scholar
  20. 20.
    Ballard, S. T., Trout, L., Garrison, J., and Inglis, S. K. (2006) Ionic mechanism of forskolin-induced liquid secretion by porcine bronchi. Am J Physiol Lung Cell Mol Physiol 290, L97–L104.PubMedCrossRefGoogle Scholar
  21. 21.
    Joo, N. S., Saenz, Y., Krouse, M. E., and Wine, J. J. (2002) Mucus secretion from single submucosal glands of pig. Stimulation by carbachol and vasoactive intestinal peptide. J Biol Chem 277, 28167–28175.PubMedCrossRefGoogle Scholar
  22. 22.
    Ianowski, J. P., Choi, J. Y., Wine, J. J., and Hanrahan, J. W. (2007) Mucus secretion by single tracheal submucosal glands from normal and cystic fibrosis transmembrane conductance regulator CFTR knock-out mice. J Physiol 580, 301–314.PubMedCrossRefGoogle Scholar
  23. 23.
    Joo, N. S., Irokawa, T., Wu, J. V., Robbins, R. C., Whyte, R. I., and Wine, J. J. (2002) Absent secretion to vasoactive intestinal peptide in cystic fibrosis airway glands. J Biol Chem 277, 50710–50715.PubMedCrossRefGoogle Scholar
  24. 24.
    Joo, N. S., Irokawa, T., Robbins, R. C., and Wine, J. J. (2006) Hyposecretion, not hyperabsorption, is the basic defect of cystic fibrosis airway glands. J Biol Chem 281, 7392–7398.PubMedCrossRefGoogle Scholar
  25. 25.
    Klockars, M., and Reitamo, S. (1975) Tissue distribution of lysozyme in man. J Histochem Cytochem 23, 932–940.PubMedCrossRefGoogle Scholar
  26. 26.
    Goetz, D. H., Holmes, M. A., Borregaard, N., Bluhm, M. E., Raymond, K. N., and Strong, R. K. (2002) The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. Mol Cell 10, 1033–1043.PubMedCrossRefGoogle Scholar
  27. 27.
    Sagel, S. D., Sontag, M. K., and Accurso, F. J. (2009) Relationship between antimicrobial proteins and airway inflammation and infection in cystic fibrosis. Pediatr Pulmonol 44, 402–409.PubMedCrossRefGoogle Scholar
  28. 28.
    Casado, B., Pannell, L. K., Iadarola, P., and Baraniuk, J. N. (2005) Identification of human nasal mucous proteins using proteomics. Proteomics 5, 2949–2959.PubMedCrossRefGoogle Scholar
  29. 29.
    Garcia-Caballero, A., Rasmussen, J. E., Gaillard, E., Watson, M. J., Olsen, J. C., Donaldson, S. H., et al. (2009) SPLUNC1 regulates airway surface liquid volume by protecting ENaC from proteolytic cleavage. Proc Natl Acad Sci USA 106, 11412–11417.PubMedCrossRefGoogle Scholar
  30. 30.
    Peden, D. B., Hohman, R., Brown, M. E., Mason, R. T., Berkebile, C., Fales, H. M., et al. (1990) Uric acid is a major antioxidant in human nasal airway secretions. Proc Natl Acad Sci USA 87, 7638–7642.PubMedCrossRefGoogle Scholar
  31. 31.
    Peden, D. B., Brown, M. E., Wade, Y., Raphael, G. D., Berkebile, C., and Kaliner, M. A. (1991) Human nasal glandular secretion of novel antioxidant activity: cholinergic control. Am Rev Respir Dis (Now the Am J Respir Crit Care Med) 143, 545–552.Google Scholar
  32. 32.
    Conner, G. E., Wijkstrom-Frei, C., Randell, S. H., Fernandez, V. E., and Salathe, M. (2007) The lactoperoxidase system links anion transport to host defense in cystic fibrosis. FEBS Lett 581, 271–278.PubMedCrossRefGoogle Scholar
  33. 33.
    Moskwa, P., Lorentzen, D., Excoffon, K. J., Zabner, J., McCray, P. B., Jr., et al (2006) A novel host defense system of airways is defective in cystic fibrosis. Am J Respir Crit Care Med 175, 174–183.PubMedCrossRefGoogle Scholar
  34. 34.
    Kesimer, M., Makhov, A. M., Griffith, J. D., Verdugo, P., and Sheehan, J. K. (2010) Unpacking a gel forming mucin: a view of MUC5B organization after granular release. Am J Physiol Lung Cell Mol Physiol 298, L15–L22.PubMedCrossRefGoogle Scholar
  35. 35.
    Wine, J. J., and Joo, N. S. (2004) Submucosal glands and airway defense. Proc Am Thorac Soc 1, 47–53.PubMedCrossRefGoogle Scholar
  36. 36.
    Quinton, P. M. (2008) Cystic fibrosis: impaired bicarbonate secretion and mucoviscidosis. Lancet 372, 415–417.PubMedCrossRefGoogle Scholar
  37. 37.
    Matsui, H., Wagner, V. E., Hill, D. B., Schwab, U. E., Rogers, T. D., Button, B., et al. (2006) A physical linkage between cystic fibrosis airway surface dehydration and Pseudomonas aeruginosa biofilms. Proc Natl Acad Sci USA 103, 18131–18136.PubMedCrossRefGoogle Scholar
  38. 38.
    Song, Y., Salinas, D., Nielson, D. W., and Verkman, A. S. (2006) Hyperacidity of secreted fluid from submucosal glands in early cystic fibrosis. Am J Physiol Cell Physiol 290, C741–C749.PubMedCrossRefGoogle Scholar
  39. 39.
    Irokawa, T., Krouse, M. E., Joo, N. S., Wu, J. V., and Wine, J. J. (2004) A “virtual gland” method for quantifying epithelial fluid secretion. Am J Physiol Lung Cell Mol Physiol 287, L784–L793.PubMedCrossRefGoogle Scholar
  40. 40.
    Joo, N. S., Krouse, M. E., Wu, J. V., Saenz, Y., Jayaraman, S., Verkman, A. S., et al. (2001) HCO(3)(-) Transport in relation to mucus secretion from submucosal glands. JOP 2, 280–284.PubMedGoogle Scholar
  41. 41.
    Ballard, S. T., Parker, J. C., and Hamm, C. R. (2006) Restoration of mucociliary transport in the fluid-depleted trachea by surface-active instillates. Am J Respir Cell Mol Biol 34, 500–504.PubMedCrossRefGoogle Scholar
  42. 42.
    Lindert, J., Perlman, C. E., Parthasarathi, K., and Bhattacharya, J. (2007) Chloride-dependent secretion of alveolar wall liquid determined by optical-sectioning microscopy. Am J Respir Cell Mol Biol 36, 688–696.PubMedCrossRefGoogle Scholar
  43. 43.
    Meyrick, B., and Reid, L. (1970) Ultrastructure of cells in the human bronchial submucosal glands. J Anat 107, 281–299.PubMedGoogle Scholar
  44. 44.
    Jacquot, J., Puchelle, E., Hinnrasky, J., Fuchey, C., Bettinger, C., Spilmont, C., et al. (1993) Localization of the cystic fibrosis transmembrane conductance regulator in airway secretory glands. Eur Respir J 6, 169–176.PubMedGoogle Scholar
  45. 45.
    Finkbeiner, W. E., Shen, B. Q., and Widdicombe, J. H. (1994) Chloride secretion and function of serous and mucous cells of human airway glands. Am J Physiol 267, L206–L210.PubMedGoogle Scholar
  46. 46.
    Choi, H. K., Finkbeiner, W. E., and Widdicombe, J. H. (2000) A comparative study of mammalian tracheal mucous glands. J Anat 197, 361–372.PubMedCrossRefGoogle Scholar
  47. 47.
    Widdicombe, J. H., Chen, L. L., Sporer, H., Choi, H. K., Pecson, I. S., and Bastacky, S. J. (2001) Distribution of tracheal and laryngeal mucous glands in some rodents and the rabbit. J Anat 198, 207–221.PubMedCrossRefGoogle Scholar
  48. 48.
    Peatfield, A. C., Barnes, P. J., Bratcher, C., Nadel, J. A., and Davis, B. (1983) Vasoactive intestinal peptide stimulates tracheal submucosal gland secretion in ferret. Am Rev Respir Dis (Now Am J Respir Crit Care Med) 128, 89–93.Google Scholar
  49. 49.
    Nadel, J. A., and Davis, B. (1980) Parasympathetic and sympathetic regulation of secretion from submucosal glands in airways. Fed Proc 39, 3075–3079.PubMedGoogle Scholar
  50. 50.
    Nadel, J. A., and Davis, B. (1978) Regulation of Na+ and Cl- transport and mucous gland secretion in airway epithelium. Ciba Found Symp 54, 133–147.PubMedGoogle Scholar
  51. 51.
    Phillips, J. E., Hey, J. A., and Corboz, M. R. (2002) Computer assisted measurement of airway gland secretions by the hillocks technique. Comput Methods Prog Biomed 68, 215–222.CrossRefGoogle Scholar
  52. 52.
    Ueki, I., German, V. F., and Nadel, J. A. (1980) Micropipette measurement of airway submucosal gland secretion. Autonomic effects. Am Rev Respir Dis (Now Am J Respir Crit Care Med) 121, 351–357.Google Scholar
  53. 53.
    German, V. F., Ueki, I. F., and Nadel, J. A. (1980) Micropipette measurement of airway submucosal gland secretion: laryngeal reflex. Am Rev Respir Dis (Now Am J Respir Crit Care Med) 122, 413–416.Google Scholar
  54. 54.
    German, V. F., Corrales, R., Ueki, I. F., and Nadel, J. A. (1982) Reflex stimulation of tracheal mucus gland secretion by gastric irritation in cats. J Appl Physiol 52, 1153–1155.PubMedGoogle Scholar
  55. 55.
    Leikauf, G. D., Ueki, I. F., and Nadel, J. A. (1984) Autonomic regulation of viscoelasticity of cat tracheal gland secretions. J Appl Physiol 56, 426–430.PubMedGoogle Scholar
  56. 56.
    Inglis, S. K., Corboz, M. R., Taylor, A. E., and Ballard, S. T. (1997) In situ visualization of bronchial submucosal glands and their secretory response to acetylcholine. Am J Physiol 272, L203–L210.PubMedGoogle Scholar
  57. 57.
    Sasaki, H., Sasaki, T., Shimura, S., and Takishima, T. (1986) Effect of fenoterol on secretions of an isolated single submucosal gland from the trachea. Respiration 50 Suppl 2, 266–269.PubMedCrossRefGoogle Scholar
  58. 58.
    Shimura, S., Sasaki, T., Sasaki, H., and Takishima, T. (1986) Contractility of isolated single submucosal gland from trachea. J Appl Physiol 60, 1237–1247.PubMedGoogle Scholar
  59. 59.
    Shimura, S., Sasaki, T., Okayama, H., Sasaki, H., and Takishima, T. (1987) Effect of substance P on mucus secretion of isolated submucosal gland from feline trachea. J Appl Physiol 63, 646–653.PubMedGoogle Scholar
  60. 60.
    Shimura, S., Sasaki, T., Okayama, H., Sasaki, H., and Takishima, T. (1987) Neural control of contraction in isolated submucosal gland from feline trachea. J Appl Physiol 62, 2404–2409.PubMedCrossRefGoogle Scholar
  61. 61.
    Shimura, S., Sasaki, T., Ikeda, K., Sasaki, H., and Takishima, T. (1988) VIP augments cholinergic-induced glycoconjugate secretion in tracheal submucosal glands. J Appl Physiol 65, 2537–2544.PubMedGoogle Scholar
  62. 62.
    Sasaki, T., Shimura, S., Ikeda, K., Sasaki, H., and Takishima, T. (1989) Platelet-activating factor increases platelet-dependent glycoconjugate secretion from tracheal submucosal gland. Am J Physiol 257, L373–L378.PubMedGoogle Scholar
  63. 63.
    Sasaki, T., Shimura, S., Sasaki, H., and Takishima, T. (1989) Effect of epithelium on mucus secretion from feline tracheal submucosal glands. J Appl Physiol 66, 764–770.PubMedGoogle Scholar
  64. 64.
    Ishihara, H., Shimura, S., Sato, M., Masuda, T., Ishide, N., Miura, M., et al. (1990) Intracellular calcium concentration of acinar cells in feline tracheal submucosal glands. Am J Physiol 259, L345–L350.PubMedGoogle Scholar
  65. 65.
    Sasaki, T., Shimura, S., Ikeda, K., Sasaki, H., and Takishima, T. (1990) Sodium efflux from isolated submucosal gland in feline trachea. Am J Physiol 258, L112–L117.PubMedGoogle Scholar
  66. 66.
    Shimura, S., Sasaki, T., Ikeda, K., Yamauchi, K., Sasaki, H., and Takishima, T. (1990) Direct inhibitory action of glucocorticoid on glycoconjugate secretion from airway submucosal glands. Am Rev Respir Dis (Now Am J Respir Crit Care Med) 141, 1044–1049.Google Scholar
  67. 67.
    Shimura, S., Sasaki, T., Ikeda, K., Ishihara, H., Sato, M., Sasaki, H., et al. (1991) Neuropeptides and airway submucosal gland secretion. Am Rev Respir Dis (Now Am J Respir Crit Care Med) 143, S25–S27.Google Scholar
  68. 68.
    Ishihara, H., Shimura, S., Satoh, M., Masuda, T., Nonaka, H., Kase, H., et al. (1992) Muscarinic receptor subtypes in feline tracheal submucosal gland secretion. Am J Physiol 262, L223–L228.PubMedGoogle Scholar
  69. 69.
    Shimura, S., Sasaki, T., Ishihara, H., Sato, M., Sasaki, H., and Takishima, T. (1992) Autonomic innervation to feline tracheal submucosal glands for mucus glycoprotein secretion. Am J Physiol 262, L15–L20.PubMedGoogle Scholar
  70. 70.
    Shimura, S. (2000) Signal transduction of mucous secretion by bronchial gland cells. Cell Signal 12, 271–277.PubMedCrossRefGoogle Scholar
  71. 71.
    Jayaraman, S., Joo, N. S., Reitz, B., Wine, J. J., and Verkman, A. S. (2001) Submucosal gland secretions in airways from cystic fibrosis patients have normal [Na+] and pH but elevated viscosity. Proc Natl Acad Sci USA 98, 8119–8123.PubMedCrossRefGoogle Scholar
  72. 72.
    Wu, D. X., Lee, C. Y., Uyekubo, S. N., Choi, H. K., Bastacky, S. J., and Widdicombe, J. H. (1998) Regulation of the depth of surface liquid in bovine trachea. Am J Physiol 274, L388–L395.PubMedGoogle Scholar
  73. 73.
    Widdicombe, J. H., Bastacky, S. J., Wu, D. X., and Lee, C. Y. (1997) Regulation of depth and composition of airway surface liquid. Eur Respir J 10, 2892–2897.PubMedCrossRefGoogle Scholar
  74. 74.
    Haxhiu, M. A., Kc, P., Moore, C. T., Acquah, S. S., Wilson, C. G., Zaidi, S. I., et al. (2005) Brain stem excitatory and inhibitory signaling pathways regulating bronchoconstrictive responses. J Appl Physiol 98, 1961–1982.PubMedCrossRefGoogle Scholar
  75. 75.
    Wine, J. J. (2007) Parasympathetic control of airway submucosal glands: central reflexes and the airway intrinsic nervous system. Auton Neurosci 133, 35–54.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Jeffrey J. Wine
    • 1
    Email author
  • Nam Soo Joo
    • 2
  • Jae Young Choi
    • 2
    • 3
  • Hyung-Ju Cho
    • 2
  • Mauri E. Krouse
    • 2
  • Jin V. Wu
    • 2
  • Monal Khansaheb
    • 2
  • Toshiya Irokawa
    • 4
  • Juan Ianowski
    • 5
  • John W. Hanrahan
    • 6
  • Alan W. Cuthbert
    • 7
  • Kim V. Tran
    • 2
  1. 1.Cystic Fibrosis Research LaboratoryStanford UniversityStanfordUSA
  2. 2.Cystic Fibrosis Research LaboratoryStanford UniversityStanfordUSA
  3. 3.Department of OtorhinolaryngologyYonsei UniversitySeoulKorea
  4. 4.Health Administration CenterTohoku UniversitySendaiJapan
  5. 5.Department of PhysiologyUniversity of SaskatchewanSaskatoonCanada
  6. 6.Department of PhysiologyMcGill UniversityMontrealCanada
  7. 7.Department of MedicineUniversity of Cambridge, Addenbrooke’s HospitalCambridgeUK

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