Respiratory and Critical Care Section, Veterans Affairs Chicago Healthcare System-Westside Division, and Department of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612
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ABSTRACT |
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Ion transport and the electric profile of distal airways of sheep lungs
were studied in a miniature polypropylene chamber with a 1-mm aperture.
Small airways with an inner diameter < 1 mm were isolated, opened
longitudinally, and then mounted as a flat sheet onto the 1-mm aperture
where it was glued and secured with an O-ring. Both sides of the tissue
were bathed with identical physiological solutions at 37°C and
oxygenated. Pooled data from 27 distal airways showed an inner airway
diameter of 854 ± 22 (SE) µm and a transepithelial potential
difference (PD) of 1.86 ± 0.29 mV, lumen negative. Short-circuit
current (Isc) was 25 ± 3.5 µA/cm2, tissue resistance was 96 ± 14 , and
conductance was 15.2 ± 1.7 mS/cm2. At baseline,
amiloride-sensitive Na transport accounted for 51% of
Isc (change in Isc = 9.7 ± 2.6 µA/cm2; n = 8 airways), corresponding to 0.36 µeq · cm
2 · h
1. Treatment
with 0.1 mM bumetanide did not reduce the Isc
(n = 5 airways). Exposure to 1 µM Ca ionophore
A-23187 raised the Isc by 9 µA/cm2
(47%; P < 0.03; n = 6 airways). The
latter effect was blunted by bumetanide. Carbachol at 1 µM provoked a
biphasic response, an initial rapid rise in Isc
followed by a decline (n = 3 airways). There was no
significant increase in PD or Isc in response to isoproterenol or dibutyryl cAMP. The data suggest that Na absorption constitutes at least 50% of baseline transport activity. Cl or other
anion secretion such as HCO3 appears to be present and
could be stimulated by raising intracellular Ca.
amiloride; bumetanide; isoproterenol; carbachol; A-23187; terminal bronchioles
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INTRODUCTION |
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STUDIES OF ION TRANSPORT ACTIVITY of airway epithelia have been largely confined to the epithelia of upper airways, namely the nasal and tracheal mucosae and the mucosa of large bronchi (9, 30). Technical difficulties in isolating and investigating the more numerous, and of great clinical relevance, distal airways (<1 mm in diameter) have hampered investigation into the physiology and pharmacology of this region of the mammalian lung. Small airways contribute over 85-90% to the total mucous membrane surface area of the conducting airways (29). Furthermore, the distal airways are the main sites of pathology in asthma (28), chronic bronchitis (12), and cystic fibrosis (16). A microperfusion method in isolated sheep distal airways was used to measure the response of transepithelial electrical potential difference (PD) to a variety of pharmacological manipulations (4, 5). Ballard and colleagues (6, 7) and Inglis et al. (20) studied porcine distal airways using the microperfusion technique and cable analysis to obtain both the short-circuit current (Isc) and the resistance (R).
Although transepithelial PD is a useful measure of ion transport rate, its magnitude could be blunted by a paracellular shunt pathway, especially in low-resistance epithelia, as well as by artifacts of edge damage. To gather a more comprehensive understanding of the electric profile, a new method was developed for mounting the distal airways of sheep as a planar sheet in a miniature chamber that allows measurement of PD in addition to Isc and tissue conductance (G) and R.
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METHODS |
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Tissue mounting chamber.
A polypropylene test tube (diameter 1.8 cm, length 9.3 cm; VWR
Scientific, Philadelphia, PA) was used for tissue mounting (Fig.
1). The rounded tube end was sanded with
no. 60 coarse sandpaper and then smoothed with no. 150 fine sandpaper
to provide a level surface on which to place the tissue. A 20.5-gauge
needle was used to puncture the tube and provide a 1.0-mm opening on
which to mount the bronchiole. The hole opening was identified with an
inert colored wax. A 7 × 1.6-cm "finger" was cut from a
powder-free latex glove, and a 3-mm hole was made at its tip where an
impermeable butyl rubber O-ring (3-mm inner diameter) was glued. A thin
layer of Sylgard (Dow-Corning, Midland, MI) was applied onto the O-ring surface facing the epithelium to ensure physical and electrical insulation of the epithelium. The mounting test tube was immersed in a
50-ml insulated glass beaker. To control the temperature at 37°C,
copper tubing connected to a circulating water bath encircled the
beaker.
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Tissue mounting. After being transferred to a chilled oxygenated Krebs-Henseleit solution (5), the caudal lobe of sheep lungs obtained from a local abattoir was placed in a wax dish and dissected distally, and 600- to 1,000-µm-diameter airways were carefully excised with as little connective tissue as possible. The airway was opened longitudinally with microscissors and transferred to a mounting stage. In preparation for tissue mounting, two small drops of cyanoacrylate glue were applied 2 mm from either side of the hole in the mounting tube. The longitudinally opened tissue was then gently placed over the hole, and a few seconds were allowed for the submucosal side of the tissue to adhere to the mounting tube. The latex sheath was quickly placed over the tube while the O-ring was pressed against the luminal surface and secured. Total mounting time was usually ~2 min. After 3 ml of Krebs-Henseleit solution were placed in the mounting tube, the tube was then positioned in the beaker bath so that the fluid levels were equal in both the mounting tube (submucosal bath) and the beaker (mucosal bath). Small-caliber polyethylene tubing connected to 5% CO2-95% O2 gas tanks was positioned in the mucosal and submucosal baths for stirring and gassing the bathing solutions. The luminal fluid was further circulated by a magnetic stirrer, which was stopped during electrical measurements because of added electrical "noise." Bathing fluids were changed once, 30 min after tissue mounting. Bath volumes were kept constant by the periodic addition of distilled water to compensate for evaporated water.
Electric parameters. Ag-AgCl voltage-sensing electrodes encased in polyethylene tubing and filled with 3 M KCl-3% agar were placed 3 mm from the submucosal side and 8-10 mm from the luminal side of the tissue. Similar current-passing electrodes were placed 15-25 mm from each side of the preparation. The chamber was tested for any bias PD between the two voltage electrodes and any part of the apparatus that may contribute to a nonbiological PD. The mounting tube was immersed before the experiment in an antistatic solution of dimethylammonium chloride to eliminate static electrical charges. The tube was placed in the beaker bath, and the resulting PD was then nullified just before the tissue was mounted. All electrodes were connected to a head stage of an epithelial voltage clamp (Warner EC-825LV, Hamden, CT). The current was further amplified with a DC amplifier/low-pass filter (Warner LPF 202; Fig. 1A).
Next, we measured the resistance of the fluid (Rf) intervening between the two voltage-sensing electrodes, including the mounting tube hole. This resistance could not be subtracted electronically because such an attempt introduced oscillations in the electronics circuitry. After measurement of Rf, the tissue was mounted, and the PD was closely monitored for stabilization, which usually was achieved in 60 min. Stabilization was reached when two PD values recorded at 5-min intervals were within 5% of each other. Once stabilization was achieved, the tissue was voltage clamped for ~45 s toExperimental protocol. After the distal airway was mounted, the PD, Isc, and Rm were recorded every 15 min until the PD stabilized (control period). A drug was then added to either the luminal or submucosal bath, and electric data were collected every 2 min for 10-15 min. After completion of the protocol, the tissue width was measured for calculation of the airway inner diameter. Finally, the tissue was removed, and the mounting tube was reassembled to obtain final data points for the system PD and the Rf. This maneuver was used to verify that these variables did not change during the course of the experiment due to electrode drift or a change in the mounting system static charges. Such changes were infrequently seen and were subtracted from the measured values.
Chemicals. Chemicals were purchased from Sigma (St. Louis, MO). In preliminary experiments, we tested the effect of the solvents used to dissolve drugs in volumes equivalent to those used in subsequent experiments and found no appreciable alterations in the electric profile (DMSO, n = 2 experiments; ethanol, n = 3 experiments; NaOH, n = 3 experiments).
Statistical analysis. The data presented are means ± SE. The significance of differences between control and experimental data was assessed by Student's t-test for paired and nonpaired data as appropriate.
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RESULTS |
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The relationship between chamber aperture and Rf
measurements.
In a uniform conductor (26), the R is directly
proportional to its length (L) and inversely proportional to
its cross-sectional area (A) as determined by
R = L/A, where
is the specific resistance or resistivity of the material such as a
salt solution at a given temperature.
has the dimensions
of resistance multiplied by length; i.e., its units are ohms times
centimeters (
· cm).
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Comparison of the electric profile of sheep tracheal mucosa mounted
in miniature versus standard Ussing chambers.
Dissection and mounting of distal airways in the miniature chamber
entails variable degrees of harsh tissue handling, which would
adversely affect the tissue electrophysiological characteristics as
well as its response to various experimental manipulations. To assess
the veracity of the miniature chamber, segments of sheep tracheal
mucosa (1, 17, 25, 27) were mounted in a standard Ussing
chamber (hole diameter 9 mm), and simultaneously, similar tracheal
segments were mounted in the miniature chamber. After baseline
electrophysiological variables were recorded, amiloride (0.1 mM) was
added to the luminal bath followed by 0.1 mM ouabain to the submucosal
bath. Results show that the baseline tracheal PD and R
values in the miniature chamber were about one-half of those in the
standard Ussing chamber (P < 0.007), whereas the Isc values were about the same in both chambers
(Table 1). In response to amiloride,
Isc declined by 68 ± 8 µA/cm2 in the miniature chamber, which was not
significantly different from a 50 ± 11 µA/cm2 fall
in the standard Ussing chamber. Ouabain further reduced the residual
Isc by 10 ± 17 µA/cm2 in the
miniature chamber and by 40 ± 12 µA/cm2 in the
standard chamber. These declines were not statistically different from
each other.
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Response of sheep distal airways to ion transport modulators.
A total of 27 distal airways were studied. The mean inner diameter was
854 ± 22 µm (range 637-1,000 µm). Transepithelial PD was
1.86 ± 0.29 mV (range 0.7-8.5 mV), lumen negative;
Isc was 25 ± 3.5 µA/cm2;
R was 96 ± 14 ; and G was 15.2 ± 1.7 mS/cm2.
Effect of amiloride.
To investigate the role of Na channels in distal airways, we added 0.1 mM amiloride to the luminal bath. Amiloride reduced the
Isc by 9.7 ± 3 µA/cm2 or
51% of baseline (Table 2).
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Effect of bumetanide. Addition of 0.1 mM bumetanide to the submucosa had no discernable effect on distal airway electric variables (n = 5 airways). Increasing the bumetanide concentration to 1 mM caused a reduction in PD from 1.5 ± 0.4 to 0.98 ± 0.2 mV (P < 0.05; n = 5 airways), without a significant change in Isc. This suggests a nonspecific effect due to the high bumetanide concentration or the solvent (DMSO).
Effect of isoproterenol and dibutyryl cAMP.
Sheep distal airways did not respond to bilateral addition of
isoproterenol with or without luminal amiloride pretreatment. Likewise,
no response was seen when both sides of the airway were exposed to
dibutyryl cAMP (Table 3).
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Response to Ca ionophore A-23187.
Changes in intracellular Ca concentration ([Ca]i) may act
as a second messenger for the regulation of Cl and Na transport in
epithelia including respiratory epithelia (3, 11, 17). The
Ca ionophore A-23187 has been shown to enhance Cl secretion, suppress
Na absorption, and promote prostaglandin release in the dog trachea
(3, 11). Luminal A-23187 significantly raised the PD and
Isc (Table
4). Pretreatment with 100 µM luminal amiloride failed to abolish the effect of A-23187
(Table 4, Fig. 4), suggesting that
A-23187 did not affect Na transport, in contrast to observations in the
sheep trachea (17). To inhibit Cl secretion, a Na-K-2Cl cotransport inhibitor, bumetanide (100 µM), was added to the luminal bath, followed 15 min later by A-23187. Bumetanide did not change the
electric profile but did blunt the effect of A-23187 (Table 4). The
data suggest that raising the [Ca]i stimulated a
Ca-dependent anion secretion such as Cl or HCO3.
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Effect of carbachol.
To stimulate cholinergic receptors, 1 µM carbachol was applied to the
luminal bath. Carbachol tended to raise the PD and
Isc for a few minutes initially, followed by a
decline below baseline values that persisted for at least 30 min (Table
5). The pattern of a biphasic response
confirms earlier observations in cannulated and perfused sheep small
airways (4).
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DISCUSSION |
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The aims of this report are twofold: first, to validate the
utility of the miniature chamber and second, to confirm and extend the
previously presented data about patterns of ion transport in sheep
distal airways (4, 5). Comparison of tracheal mucosal segments mounted in the standard Ussing chamber with segments mounted
in the miniature chamber reveals that their Isc
values were comparable, but baseline PD and R values in the
miniature chamber were about one-half of those in the Ussing chamber
(Table 1). This suggests that these differences are likely due to edge damage in the miniature chamber because of the high ratio of
circumference to surface area (18) compared with this
ratio in tissues mounted in the standard 9-mm Ussing chambers. Another
possible source of measurement error is the uncertainty related to the
current pathway through the mounted tissue as it traverses across the 0.785-mm2 submucosal aperture and the much larger
7.065-mm2 luminal surface area demarcated by the inner
circumference of the O-ring. In preliminary experiments with sheep
tracheae in the miniature chamber, the conclusion was reached that the
area across which the current passes through the surface epithelium had
to be calculated on the basis of a luminal surface area delimited by
the O-ring rather than by the 1-mm aperture facing the submucosal side
of the tissue (Fig. 1B). It is likely that the current
passed through a cone-shaped thickness of the tissue rather than
through a disk-shaped thickness. The ratio of Rt
(~96 ) to the system resistance (~800
) was a low 12% and
therefore might have introduced another source of inaccuracy.
In the short-circuited sheep tracheal mucosa, 30% of Isc is due to HCO3 and Cl secretion and Na-glucose cotransport (1, 17). The remaining 70% of the Isc is attributed to isolated Na absorption, with the amiloride-sensitive component constituting 40% of the Isc and the amiloride-insensitive portion comprising 30% of the Isc. Data presented in Table 1 show that amiloride applied to the sheep trachea reduced the Isc by 46% in the standard Ussing chamber, a proportion not appreciably different from that observed by Graham et al. (17). However, a larger proportion of the Isc (67%) was reduced by amiloride in sheep tracheae mounted in the miniature chamber. The importance of this apparent difference in the magnitude of amiloride-sensitive Na transport is not clear because it did not achieve significance. Attempts to stimulate Isc with isoproterenol or cAMP failed to do so in tracheae mounted in either the Ussing chamber or the miniature chamber. This observation is in agreement with those of Graham et al., where forskolin together with the phosphodiesterase inhibitor zardaverine produced only a transient 6% increase in Isc. The data suggest that tissues mounted in the miniature chambers retained Na transport machinery, but it is not clear if other transport processes are affected.
In this paper, as in previous publications (4, 5), small airways with an inner diameter of 1 mm or less were used. Light microscopy revealed neither submucosal glands nor cartilage; however, Clara cells could not be detected convincingly (5, 22). Based on these observations, it is estimated that the airways used in this and earlier reports (22, 24) represent the 24th to 27th lobular bronchi and terminal bronchioles just before they bifurcate into the respiratory bronchioles. Comparison of electric PD of microperfused sheep distal airways with those in the miniature chamber presented in this report shows that the baseline PD, on average, was higher in the microperfused preparation than in the planar sheet in the miniature chamber (3.52 vs. 1.86 mV) (4). The latter preparation also was not responsive to cAMP or isoproterenol (4, 5). However, in both preparations, luminal amiloride caused comparable decline in PD (65 vs. 51%). These comparisons suggest that the planar sheet preparation retains the basic characteristics of distal airways but is less robust than the intact tissue. This should not detract from the usefulness of the miniature chamber method, yet it calls for further refinement of methods to reduce tissue damage during dissection and mounting. The planar sheet preparation has an advantage over the microperfused airways in that it provides a more lucid understanding of the distal airway response to pharmacological agents with alterations in the Isc and tissue conductance as well as in the PD.
The amiloride-sensitive Na transport constituted 51% of the basal
Isc and carried an average current of 9.7 µA/cm2, corresponding to 0.36 µeq · cm2 · h
1, as the
minimal value for resting Na absorption in sheep distal airways. The
remaining 49% of Isc could represent
amiloride-insensitive Na absorption and other electrogenic processes.
A-23187 raises [Ca]i and is therefore used to investigate the role of this rise on ion channel activities. This agent elicited a significant increase in the Isc in the presence and absence of amiloride-sensitive Na absorption (Table 4). This observation suggests that A-23187 stimulated anion secretion, possibly Cl or HCO3 secretion, especially because preincubation with bumetanide eliminated the effect of A-23187 (Table 4). In contrast to this observation, Inglis et al. (20) reported that in the porcine distal bronchi, A-23187 did not alter the Isc but increased the PD and Rt, which suggests that it did not affect active ion transport but increased the paracellular resistance pathway.
To explore the role of cholinergic receptors on ion transport in the distal airways, the muscarinic agonist carbachol was used. Carbachol releases Ca from intracellular stores but does not raise cAMP (15). In neuroblastoma hybrid cells (19), carbachol has been shown to cause breakdown of membrane phosphatidylinositol 4,5-bisphosphate into phosphatidylinositol 1,4,5-trisphosphate and diacylglycerol. The latter then activates protein kinase C, causing phosphorylation of Ca-activated K channels (14, 15). In the colonic cell line T84, it was found that cholinergic stimulation with carbachol caused increased basolateral K conductance (15). The K efflux hyperpolarizes the cell and increases the driving force for Cl exit across the apical membrane. Devor et al. (14) used patch-clamped T84 cells and confirmed these findings. They reported that carbachol induced an oscillating Ca-activated K conductance, with no Cl conductance. Sheep distal airways mounted in the miniature chamber responded to carbachol by an initial rise followed by a fall in the PD and Isc (Table 5). This pattern is similar to what was noted previously in the microperfused preparation (4) where Cl removal or exposure to the apical Cl channel inhibitor diphenylamine-2-carboxylate did not abolish the airway response to carbachol. The observations are in agreement with the reported effects of carbachol on the basolateral Ca-activated K channels. In porcine distal bronchi (3.62-mm diameter), submucosal acetylcholine increased PD and Isc and was thought to induce both Cl and HCO3 secretion (20). In contrast to the effect of carbachol on sheep distal airways, mucosal exposure of canine tracheae to acetylcholine in vivo or in vitro does not alter the PD (10, 23) but causes the electroneutral secretion of Na and Cl, most likely from the submucosal glandular epithelium (8). Cholinergic stimulation of ferret tracheae with submucosal methacholine augmented albumin, lysozyme, and fluid secretions (13). The stimulation of parasympathetic nerves and the application of cholinergic agonists stimulate submucosal glands (8). Sheep distal bronchi beyond the 25th generation lack submucosal glands (22); therefore, the effect of carbachol could be attributed to the bronchiolar surface epithelium.
In summary, this report presents physiological observations on a newly devised miniature method for studying terminal bronchioles, including human distal airways (2). Evidence is provided that sheep terminal bronchioles have the potential for Na absorption and anion secretion. The latter is sensitive to changes in [Ca]i. Muscarinic receptor stimulation appears to promote anion secretion, probably by increasing the conductance of Ca-activated K channels.
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ACKNOWLEDGEMENTS |
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These studies were supported by a Merit Review Award from the Department of Veterans Affairs.
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FOOTNOTES |
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Address for reprint requests and other correspondence: F. J. Al-Bazzaz, VA Chicago Healthcare System-Westside Division, Medical Service (MP111), 820 S. Damen Ave., Chicago, IL 60612 (E-mail: faiq.al-bazzaz{at}med.va.gov).
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.
Received 9 July 1999; accepted in final form 8 June 2001.
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