Airway surface liquid pH in well-differentiated airway epithelial cell cultures and mouse trachea

Airway surface liquid (ASL) pH hasbeen proposed to be important in the pathophysiology of cysticfibrosis, asthma, and cough. Ratio image analysis was used to measurepH in the ASL after staining with the fluorescent pH indicator2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-dextran. ASL pH in bovine airway cell cultures grown at anair-liquid interface was 6.98 ± 0.06 in the absence and 6.81 ± 0.04 in the presence of HCO/CO₂. Steady-state ASL pH changed in parallel to changes in bath pH and wasacidified by Na⁺ or Cl replacement but wasnot affected by the inhibitors amiloride, glibenclamide, or4,4'-dinitrostilbene-2,2'-disulfonic acid. In response to suddenacidification or alkalization of the ASL by ~0.4 pH units byHCl/NaOH, ASL pH recovered to its initial value at a rate of 0.035 pHunits/min (HCO) and 0.060 pH units/min(+HCO); the pH recovery rate was reduced byamiloride and H₂DIDS. In anesthetized mice in which thetrachea was surgically exposed for measurement of BCECF-dextranfluorescence through the translucent tracheal wall, ASL pH was7.14 ± 0.01. ASL pH was sensitive to changes in blood pH createdby metabolic (HCl or NaHCO₃ infusion) or respiratory (hyperventilation, hypoventilation) mechanisms. ASL pH is thus primarily determined by basolateral fluid pH, andH⁺/OH transport between the ASL andbasolateral fluid involves amiloride-sensitive Na⁺/H⁺ exchange and stilbene-sensitiveCl/HCO exchange. The rapid response ofASL pH to changes in systemic acid-base status may contribute to airwayhypersensitivity in asthma and other airway diseases.

     

Estimation of thickness of airwaysurface liquid in ferret trachea in vitro

Duneclift, S., U. Wells, and J. Widdicombe. Estimationof thickness of airway surface liquid in ferret trachea in vitro. J. Appl. Physiol. 83(3): 761-767, 1997.The tracheae of ferrets and rabbits were mounted in vitro inorgan baths. While the tracheae were liquid filled, the permeabilitycoefficient ( P) was determined, and then while thetracheae were air filled, the percent clearance for^99mTc-labeleddiethylenetriaminepentaacetic acid (DTPA) was determined. The thicknessof airway surface liquid (ASL) was estimated by three methods.1) The initial concentration of^99mTc-DTPA and the total amount of^99mTc-DTPA (the sum of thatentering the outside medium, that draining from the trachea, and thatwashed out at the end of 40 min) gave the initial volume of ASL andthus its thickness. Mean values were 45.7 µm for the ferret and 41.9 µm for the rabbit. 2) Estimates ofASL thickness at the end of the 40-min period, based on the final^99mTc-DTPA concentration and theamount in the washout, were 42.9 µm for ferret and 45.4 µm forrabbit. 3) The ratio of Pto percent clearance gave mean ASL thickness values of 49.2 µm forthe ferret and 40.3 µm for the rabbit. Thus three separate methodsfor determining ASL thickness give very similar results, with means inthe range 40-49 µm. Administration of methacholine or atropineto ferret tracheae did not significantly change ASL thickness.

     

Role of intrinsic airway neurons in ozone-induced airway hyperresponsiveness in ferret trachea.

Exposure to ozone (O(3)) enhances airway responsiveness, which is mediated partly by the release of substance P (SP) from airway neurons. In this study, the role of intrinsic airway neurons in O(3)-induced airway responses was examined. Ferrets were exposed to 2 ppm O(3) or air for 1 h. Reactivity of isolated tracheal smooth muscle to cholinergic agonists was significantly increased after O(3) exposure, as were contractions to electrical field stimulation at 10 Hz. Pretreatment with CP-99994, a neurokinin type 1 receptor antagonist, partially abolished the O(3)-induced reactivity to cholinergic agonists and electrical field stimulation. The O(3)-enhanced airway responses were present in tracheal segments cultured for 24 h, a procedure shown to deplete sensory nerves while maintaining viability of intrinsic airway neurons, and all the enhanced smooth muscle responses were also diminished by CP-99994. Immunocytochemistry showed that the percentage of SP-containing neurons in longitudinal trunk and the percentage of neurons innervated by SP-positive nerve fibers in superficial muscular plexus were significantly increased at 1 h after exposure to O(3). These results suggest that enhanced SP levels in airway ganglia contribute to O(3)-induced airway hyperresponsiveness.     

Chloride transport in the ferret trachea

A net secretion of chloride stimulated by carbamylcholine was observed in whole trachea. Luminal anthracene-9-carboxylic acid inhibited the net secretion of chloride and the transepithelial potential difference across the isolated trachea. Submucosal ouabain inhibited the net secretion of chloride and submucosal ouabain, or submucosal bumetanide inhibited the transepithelial potential difference across the isolated trachea. Short-circuited flat sheets of trachea manifested a net secretion of chloride induced by carbamylcholine. Serosal ouabain inhibited the short-circuit current and net chloride flux across isolated flat sheets of trachea.     

Substance P released from intrinsic airway neurons contributes to ozone-enhanced airway hyperresponsiveness in ferret trachea.

Exposure to ozone (O3) induces airway hyperresponsiveness mediated partly through the release of substance P (SP) from nerve terminals in the airway wall. Although substantial evidence suggests that SP is released by sensory nerves, SP is also present in neurons of airway ganglia. The purpose of this study was to investigate the role of intrinsic airway neurons in O3-enhanced airway responsiveness in ferret trachea. To remove the effects of sensory innervation, segments of ferret trachea were maintained in culture conditions for 24 h before in vitro exposure to 2 parts/million of O3 or air for 1 h. Sensory nerve depletion was confirmed by showing that capsaicin did not affect tracheal smooth muscle responsiveness to cholinergic agonist or contractility responses to electrical field stimulation (EFS). Contractions of isolated tracheal smooth muscle to EFS were significantly increased after in vitro O3 exposure, but the constrictor response to cholinergic agonist was not altered. Pretreatment with CP-99994, an antagonist of the neurokinin 1 receptor, attenuated the increased contraction to EFS after O3 exposure but had no effect in the air exposure group. The number of SP-positive neurons in longitudinal trunk ganglia, the extent of SP innervation to superficial muscular plexus nerve cell bodies, and SP nerve fiber density in tracheal smooth muscle all increased significantly after O3 exposure. The results show that release of SP from intrinsic airway neurons contributes to O3-enhanced tracheal smooth muscle responsiveness by facilitating acetylcholine release from cholinergic nerve terminals.     

Effects of airway surface liquid pH on host defense in cystic fibrosis

Cystic fibrosis is a lethal genetic disorder characterized by viscous mucus and bacterial colonization of the airways. Airway surface liquid represents a first line of pulmonary defense. Studies in humans and animal models of cystic fibrosis indicate that the pH of airway surface liquid is reduced in the absence of cystic fibrosis transmembrane conductance regulator function. Many aspects of the innate host defense system of the airways are pH sensitive, including antimicrobial peptide/protein activity, the rheological properties of secreted mucins, mucociliary clearance, and the activity of proteases. This review will focus on how changes in airway surface liquid pH may contribute to the host defense defect in cystic fibrosis soon after birth. Understanding how changes in pH impact mucosal immunity may lead to new therapies that can modify the airway surface liquid environment, improve airway defenses, and alter the disease course.This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.     

Postpneumonectomy airway growth in the ferret

To investigate the participation of the conducting airways in compensatory growth following partial lung resection, bronchial casts of six ferrets having undergone right-sided pneumonectomy at 8 wk of age were compared with those of five sham-operated control animals. At maturity, the left lungs of the postpneumonectomy animals were 65% larger than those of the controls. Central airway cross-sectional areas at 10 specific locations in each cast were 12% larger in the postpneumonectomy animals compared with controls. To characterize the size of more peripheral airways, the size and number of the terminal bronchioles subtended by each airway in each left lower lobe cast were identified so that the fraction of the lobe served by that airway could be estimated. The characteristic cross-sectional areas of airway serving 0.7, 2.2, and 9.5% of the left lower lobe in postpneumonectomy animals were 18, 13, and 13% larger than those of controls, but this difference was statistically significant only at the two more peripheral levels. Although airway areas were larger in postpneumonectomy animals, the ratio of airway cross-sectional area to the 0.67 power of lung volume was 20-26% smaller in operated than in control animals at each of the four levels. Following pneumonectomy in the weanling ferret, central and peripheral conducting airways increase in cross-sectional area to similar degrees, but this airway growth is less than the compensatory increase in lung volume.     

Somatostatin potentiates cholinergic neurotransmission in ferret trachea

We studied the effect of somatostatin on contractile responses to electrical field stimulation (EFS) in isolated ferret tracheal segments. Somatostatin (up to 10(-5) M) did not change resting tension, but it potentiated the contractile response to EFS dose dependently, with a maximum effect at 10(-6) M. Thus, at a concentration of 10(-6) M, somatostatin significantly decreased the mean log of EFS frequency producing 50% of maximum contraction from a control value of 0.52 +/- 0.07 to 0.24 +/- 0.06 (SE) Hz (P less than 0.01). The potentiating effect of somatostatin (10(-6) M) was not inhibited by hexamethonium, indomethacin, BW755C, pyrilamine, methysergide, or D,Pro2,D,Trp7,9-SP, but it was inhibited by atropine or by the somatostatin antagonist cyclo[7-aminoheptanoyl-Phe-D-Trp-Lys-Thr(Bzl)]. In contrast to EFS-induced contraction, contractions produced by acetylcholine (10(-9) to 10(-3) M) were not affected by somatostatin at a concentration of 10(-6) M. These results suggest that somatostatin potentiates contractions produced by EFS via presynaptic cholinergic mechanisms and probably through a specific somatostatin receptor.     

Development of mucociliary transport in the postnatal ferret trachea.

Little is known of the developmental aspects of mucociliary transport. Previous studies have documented that newborn ferret trachea has very few ciliated cells but numerous immature secretory cells in the epithelium and only rudimentary submucosal glands. Rapid and complete maturation occurs in the first postnatal month. This study examines mucociliary transport during this period of rapid maturation. We made direct observations of particle movement across the epithelium of ferret tracheas. No mucus transport could be demonstrated on the first day of life. Transport was discernible, although sporadic and slow, by 7 days and reached adult levels (10.7 +/- 3.7 mm/min) by 28 postnatal days. The emergence of transport capability correlated well with previously described developmental changes in ciliation, mucus secretion, and ion permeability and transport. Threshold mucus transport occurred at 1 wk of age when 20-25% of the surface cells are ciliated. The neonatal ferret appears to be a useful model for assessing integrated epithelial structure-function relationships that are important not only during early development but also during repair after airway injury involving deciliation.     

X-ray microanalysis of airway surface liquid in the mouse

The ionic composition of airway surface liquid (ASL) has been debated, and, in particular for the mouse, a wide range of values has been published. Two techniques were developed to measure the elemental composition of the ASL. X-ray microanalysis of ASL was carried out at low temperature on trachea removed from isoflurane-anesthetized animals and shock-frozen. In the second technique, dextran beads were placed on top of the epithelium of the trachea removed from pentobarbital-anesthetized animals, left to equilibrate with the ASL, dried, and subjected to X-ray microanalysis. Both techniques showed that mouse tracheal ASL has significantly lower concentrations of Na and Cl (approximately 60-80 mM) than serum. Differences between the two techniques were due to different sampling of mucus. CFTR(-/-) mice had significantly higher concentrations of Na and Cl in their ASL than age-matched controls. Pilocarpine or isoproterenol stimulation significantly reduced the ion concentrations in tracheal ASL. ASL was also collected with the dextran bead method from the nasal cavity in situ in pentobarbital-anesthetized animals. In control animals, the elemental composition of nasal fluid was similar to that of tracheal ASL. Pilocarpine stimulation caused a significant increase in Na, Cl, and K; stimulation with isoproterenol or phenylephrine caused a significant increase only in K. It is concluded that mouse ASL under unstimulated conditions is hypotonic, which may be related to the relative paucity of submucosal glands in the mouse trachea.     

Sodium and chloride concentrations, pH, and depth of airway surface liquid in distal airways

The composition and depth of the airway surface liquid (ASL) are key parameters in airway physiology that are thought to be important in the pathophysiology of cystic fibrosis and other diseases of the airways. We reported novel fluorescent indicator and microscopy methods to measure [Na+], [Cl-], pH, and depth of the ASL in large airways (Jayaraman, S., Y. Song, L. Vetrivel, L. Shankar, and A.S. Verkman. 2001. J. Clin. Invest. 107:317-324.). Here we report a stripped-lung preparation to measure ASL composition and depth in small distal airways. Distal ASL was stained with ion- or pH-sensitive fluorescent indicators by infusion into mouse trachea of a perfluorocarbon suspension of the indicator. After stripping the pleura and limited microdissection of the lung parenchyma, airways were exposed for measurement of ASL [Na+], [Cl-], and pH by ratio imaging microscopy, and depth by confocal microscopy. The stripped-lung preparation was validated in stability and tissue viability studies. ASL [Na+] was 122 +/- 2 mM, [Cl-] was 123 +/- 4 mM and pH was 7.28 +/- 0.07, and not dependent on airway size (<100- to >250-mum diameter), ENaC inhibition by amiloride, or CFTR inhibition by the thiazolidinone CFTRinh-172. ASL depth was 8-35 mum depending on airway size, substantially less than that in mouse trachea of approximately 55 mum, and not altered significantly by amiloride. These results establish a novel lung preparation and fluorescence approach to study distal airway physiology and provide the first data on the composition and depth of distal ASL.     

Proinflammatory mediators disrupt glucose homeostasis in airway surface liquid

The glucose concentration of the airway surface liquid (ASL) is much lower than that in blood and is tightly regulated by the airway epithelium. ASL glucose is elevated in patients with viral colds, cystic fibrosis, chronic obstructive pulmonary disease, and asthma. Elevated ASL glucose is also associated with increased incidence of respiratory infection. However, the mechanism by which ASL glucose increases under inflammatory conditions is unknown. The aim of this study was to investigate the effect of proinflammatory mediators (PIMs) on the mechanisms governing airway glucose homeostasis in polarized monolayers of human airway (H441) and primary human bronchial epithelial (HBE) cells. Monolayers were treated with TNF-α, IFN-γ, and LPS during 72 h. PIM treatment led to increase in ASL glucose concentration and significantly reduced H441 and HBE transepithelial resistance. This decline in transepithelial resistance was associated with an increase in paracellular permeability of glucose. Similar enhanced rates of paracellular glucose flux were also observed across excised trachea from LPS-treated mice. Interestingly, PIMs enhanced glucose uptake across the apical, but not the basolateral, membrane of H441 and HBE monolayers. This increase was predominantly via phloretin-sensitive glucose transporter (GLUT)-mediated uptake, which coincided with an increase in GLUT-2 and GLUT-10 abundance. In conclusion, exposure of airway epithelial monolayers to PIMs results in increased paracellular glucose flux, as well as apical GLUT-mediated glucose uptake. However, uptake was insufficient to limit glucose accumulation in ASL. To our knowledge, these data provide for the first time a mechanism to support clinical findings that ASL glucose concentration is increased in patients with airway inflammation.     

Modulation of cholinergic neurotransmission by vasoactive intestinal peptide in ferret trachea

We studied the effect of vasoactive intestinal peptide (VIP) on the contractile responses to electrical field stimulation (EFS) in isolated ferret tracheal segments. VIP did not change resting tension up to 2 X 10(-7) M, but it showed a biphasic effect on the responses to EFS. In concentrations up to 10(-9) M, VIP potentiated the response; at higher concentrations VIP reduced responses. Thus, at a concentration of 10(-9) M, VIP decreased the mean (+/- SE) log EFS frequency, producing 50% of maximum contraction significantly from a control value of 0.476 +/- 0.062 to 0.214 +/- 0.057 Hz (P less than 0.01); at a concentration of 2 X 10(-7) M VIP increased the half-maximal frequency from a control value of 0.513 +/- 0.086 to 0.752 +/- 0.053 Hz (P less than 0.05). The potentiating effect of VIP (10(-9) M) was not inhibited by hexamethonium, indomethacin, pyrilamine, methysergide, or [D-Pro2,D-Trp7,9] substance P. The inhibitory effect of VIP (2 X 10(-7) M) was also not inhibited by hexamethonium, indomethacin, or naloxone. In contrast to EFS-induced contraction, contractions produced by acetylcholine (10(-9) to 10(-3) M) were not affected by VIP at concentrations of 10(-9) and 2 X 10(-7) M. These results suggest that VIP modulates contractions produced by EFS via presynaptic cholinergic mechanisms and probably through a specific VIP receptor.     

Secretory phospholipases A2 stimulate mucus secretion, induce airway inflammation, and produce secretory hyperresponsiveness to neutrophil elastase in ferret trachea

Secretory phospholipases A(2) (sPLA(2)) are increased in the bronchoalveolar lavage fluid of patients with asthma and acute respiratory distress syndrome. Intratracheal sPLA(2) instillation induces acute lung injury in the rat and guinea pig. We hypothesized that sPLA(2) would stimulate mucus secretion in vitro and that intratracheal sPLA(2) exposure would induce mucus hypersecretion and airway inflammation in the ferret trachea in vivo. In vitro, porcine pancreatic sPLA(2) at a concentration of 0.5 or 5 U/ml significantly increased mucous glycoconjugate (MG) secretion from the excised ferret trachea. P-bromophenacylbromide (a sPLA(2) inhibitor), quercetin (a lipoxygenase inhibitor), or MK-886 (a 5-lipoxygenase inhibitor), each at 10(-4) M, significantly reduced sPLA(2)-induced MG secretion. sPLA(2)-stimulated MG secretion was decreased in Ca(2+)-free medium. In vivo, ferrets were intubated for 30 min once per day for 3 days using an ETT coated with 20 units of porcine pancreatic sPLA(2) mixed in water-soluble jelly. Constitutive MG secretion increased 1 day after sPLA(2) exposure and returned to control 5 days later. Human neutrophil elastase (HNE) at 10(-8) M increased MG secretion in the sPLA(2)-exposed trachea compared with that in the control trachea, but methacholine at 10(-7) M did not. sPLA(2)-induced secretory hyperresponsiveness continued for at least 5 days after sPLA(2) exposure ended. sPLA(2) increased tracheal inflammation, MG secretion, and secretory hyperresponsiveness to HNE probably through enzymatic action rather than by activation of its receptor.