Optical method for quantifying rates of mucus secretion from single submucosal glands

Joo, Nam Soo; Wu, Jin V.; Krouse, Mauri E.; Sáenz, Yamil; Wine, Jeffrey J.

doi:10.1152/ajplung.2001.281.2.l458

Cited by 78 publications

(121 citation statements)

References 37 publications

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“…A small piece of trachea was cut along the trachealis muscle, and the airway submucosa, containing the glands, was dissected from the cartilage as described previously (40). This submucosa preparation was placed in a custom-built chamber with the serosal side bathed in Krebs solution containing 1 μM indomethacin.…”

Section: Methodsmentioning

confidence: 99%

Pseudomonas aeruginosa triggers CFTR-mediated airway surface liquid secretion in swine trachea

Luan

Campanucci

Nair

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Significance Cystic fibrosis (CF) is a genetic disorder caused by mutations in the gene encoding for the anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Several organs are affected in CF, but most of the morbidity and mortality comes from lung disease caused by the failure to clear bacteria. Bacterial clearance depends on a layer of airway surface liquid (ASL) covering the airways, rich in antimicrobial compounds and mucins, that removes bacteria from the airway through mucociliary clearance. This study provides the first demonstration that inhalation of bacteria triggers CFTR-dependent ASL secretion. We suggest that this response to inhaled pathogens is an important but previously unknown part of the innate immune response that would be missing in CF patients, resulting in reduced bacterial killing and facilitating infection.

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Section: Methodsmentioning

confidence: 99%

Pseudomonas aeruginosa triggers CFTR-mediated airway surface liquid secretion in swine trachea

Luan

Campanucci

Nair

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…To minimize endogenously generated prostaglandins during tissue preparation, 1.0 M 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 (4,18,19). 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.…”

Section: Methodsmentioning

confidence: 99%

“…1A, while carbachol produced a good response (sustained carbachol secretion rates: 5.38 Ϯ 1.08 nl/min for 8 glands). 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 (4,18,19) 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.…”

Section: Confirmation Of Minimal Responses Of Cf Glands Stimulated Withmentioning

confidence: 99%

Hyposecretion, Not Hyperabsorption, Is the Basic Defect of Cystic Fibrosis Airway Glands

Joo

Irokawa

Robbins

et al. 2006

Journal of Biological Chemistry

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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 nonKunitz 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.

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“…Serial color images were captured by a CCD camera interfaced to an image analysis system. Freshly secreted fluid droplets from individual gland openings were identified and imaged after intraperitoneal pilocarpine administration, similar to the methodology for studies of submucosal gland fluid in excised fragments of porcine (15,16) and human (8) airways. The rate of volume secretion from individual submucosal gland openings was computed from the time course of increasing fluid droplet diameter assuming semi-spherical droplet geometry.…”

Section: Methodsmentioning

confidence: 99%

Aquaporin-5 Dependent Fluid Secretion in Airway Submucosal Glands

Song¹,

Verkman

2001

Journal of Biological Chemistry

185

125

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Submucosal glands in mammalian airways secrete a mixture of water, ions, and macromolecules onto the airway surface.The composition of the airway surface liquid (ASL), 1 the thin layer of fluid coating the airway epithelium, is determined in large part by the composition of fluids secreted by submucosal glands (1-3). Submucosal gland function is thought to play an important role in antimicrobial defense and in the regulation of ASL volume and composition (3-5). Abnormally viscous gland secretions in cystic fibrosis have been proposed to promote bacterial adhesion and inhibit bacterial clearance by impeding ciliary function (6 -8).Submucosal glands consist of serous tubules where active salt secretion into the gland lumen creates a small osmotic gradient driving water transport across a presumably highly water permeable epithelium. Salt secretion by serous epithelial cells is believed to involve Cl Ϫ transport by the cystic fibrosis transmembrane conductance regulator protein (CFTR), the protein which is defective in cystic fibrosis (9, 10). The glands also contain mucous cells and tubules where viscous glycoproteins are secreted. The fluid then passes through a collecting duct containing ciliated epithelial cells and onto the airway surface. Based on evidence that aquaporins facilitate nearisosmolar fluid transport in kidney proximal tubule (11) and salivary gland (12), we postulated that aquaporins and high water permeability are required for near-isosmolar fluid secretion by the epithelium of airway submucosal glands.The purpose of this study was to determine whether aquaporin water channels facilitate fluid secretion from airway submucosal glands. Aquaporin water channels AQP4 and AQP5 were immunolocalized to the basolateral and luminal membranes, respectively, of submucosal gland serous epithelial cells in mouse nasopharynx and upper airways. Methodology was developed to quantify the volume and composition of fluid secreted from submucosal glands in living mice. Comparative measurements done on wild type mice and transgenic knockout mice lacking AQP4 and AQP5 indicated an important role for AQP5 in submucosal gland fluid secretion. AQP5 is thus a rate-limiting barrier in gland fluid secretion, and so regulation of its activity may alter gland fluid secretion in cystic fibrosis and other airway diseases. EXPERIMENTAL PROCEDURESTransgenic Mice-Knockout mice deficient in AQP4 and AQP5 in a CD1 genetic background were generated by targeted gene disruption (13,14). Measurements were done in litter-matched mice (age 8 -10 weeks, body weight 20 -25 grams) produced by intercrossing of heterozygous mice. The investigators were blinded to genotype information in all measurements. Protocols were approved by the U.C.S.F. Committee on Animal Research.Measurement of Total Submucosal Gland Fluid Secretion RateMice were anesthetized with pentobarbital (50 mg/kg). After exposure

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Optical method for quantifying rates of mucus secretion from single submucosal glands

Cited by 78 publications

References 37 publications

Pseudomonas aeruginosa triggers CFTR-mediated airway surface liquid secretion in swine trachea

Pseudomonas aeruginosa triggers CFTR-mediated airway surface liquid secretion in swine trachea

Hyposecretion, Not Hyperabsorption, Is the Basic Defect of Cystic Fibrosis Airway Glands

Aquaporin-5 Dependent Fluid Secretion in Airway Submucosal Glands

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