Children's Hospital Oakland Research Institute, Oakland 94609; Lawrence Berkeley Laboratory, University of California, Berkeley 94720; and Cardiovascular Research Institute, University of California, San Francisco, California 94143
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ABSTRACT |
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The luminal surface of airways is lined by a thin film of airway surface liquid (ASL). Physiological regulation of the depth of ASL has not been reported previously. In this paper, we have used low-temperature scanning electron microscopy of rapidly frozen specimens of bovine tracheal epithelium to demonstrate alterations in the depth of ASL in response to the cholinergic agonist methacholine. We first established that methacholine selectively stimulated airway glands, with maximal secretion at ~2 min and a return to baseline within ~5 min. A 2-min exposure to methacholine increased the depth of ASL from 23 to 78 µm. Thereafter, depth decreased linearly with time, reaching 32 µm at 30 min. The initial increase in depth was blocked by bumetanide, an inhibitor of active chloride secretion, whereas the slow decline back to baseline was inhibited by amiloride, a blocker of active sodium absorption. We conclude that the methacholine-induced changes in ASL depth reflect transient gland secretion followed by liquid absorption across the surface epithelium.
low-temperature scanning electron microscopy; epithelial ion transport
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INTRODUCTION |
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AIRWAY EPITHELIUM is ciliated, and the luminal surface of the airways is lined with liquid. Previous microscopic studies of rapidly fixed specimens have suggested that this liquid is in two phases, with a mucous gel lying over a periciliary sol (15, 17, 21, 34). It is believed that low viscosity of the underlying sol permits ciliary motion, and the tips of the cilia claw at the underside of the gel and propel it toward the mouth (24). In this way, the airway surface is kept clean.
The combined depth of the sol and gel as seen in microscopic sections varies from ~5 (i.e., the length of the shortest cilia) to ~20 µm (16, 17, 21, 34). However, shrinkage artifacts due to chemical fixation, dehydration, and freeze-drying have prevented reliable estimates of the combined depth of these layers. In living tissues, estimates of airway surface liquid (ASL) depth vary from 15 µm in cultures of dog tracheal epithelium (12) to 35-50 µm for sheep trachea in vitro (22), to 87 µm for guinea pig trachea in vivo (20), and to ~200 µm for guinea pig trachea in vitro (19).
The question of whether ASL depth is under physiological control has not been addressed by microscopic studies of fixed tissues. However, in living tissues in vitro, Seybold et al. (22) failed to see changes in ASL depth in response to either methacholine or epinephrine. This result is surprising given that ion transport and the consequent fluid movements (7) across both surface and gland epithelia are under adrenergic and cholinergic control (11, 14, 18, 30).
In this paper, we have applied a new approach to studying the physiological regulation of ASL depth. Sheets of airway epithelium were rapidly frozen. While in the frozen, hydrated state, they were fractured perpendicular to the airway surface, and the depth of ASL was determined by low-temperature scanning electron microscopy. We demonstrate alterations in the depth of ASL in response to both submucosal gland secretion and active transport of sodium across the surface epithelium.
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MATERIALS AND METHODS |
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Bovine tracheas were obtained from a nearby slaughterhouse. In some
cases, within minutes of removal from the animal, pieces of trachea
were rapidly frozen by immersion in liquid nitrogen. Other tracheas
were transported to the laboratory in ice-cold, preoxygenated
Krebs-Henseleit solution (KHS) of the following composition (in mM):
120 NaCl, 25 NaHCO3, 5 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4,
6 glucose, and 0.05 phenol red. The trip from the slaughterhouse took
1-2 h. Once in the laboratory, epithelial sheets (~25
cm2) were dissected away from
the underlying tissues and cut into individual
1-cm2 squares. These were rinsed
briefly in KHS with or without amiloride. They were then pinned out,
mucosal side up, on sponges saturated with KHS and maintained in a warm
(37°C) humidified atmosphere of 95%
O2-5%
CO2. The KHS in the sponges
contained methacholine alone
(105 M), methacholine plus
amiloride (10
5 M),
methacholine plus bumetanide
(10
4 M), or no drugs. After
set periods, the tissues were rapidly frozen when a cryoprobe with a
flat polished copper surface, precooled to the temperature of liquid
nitrogen (
196°C), was brought into contact with the mucosal
surface. From this point on, the samples were maintained at less than
180°C and processed as previously described for rapidly
frozen pieces of lung (3). First, liquid nitrogen was poured over the
probe tip and adjacent tissue. The frozen tissue was then stored in
liquid nitrogen. Later, the tissue was trimmed under liquid nitrogen
with a circular dental saw and then fitted into a miniature vise with
its serosal surface facing one jaw and its mucosal surface facing the
other. Otherwise, orientation of tissue sheets within the vise was
completely random (i.e., there was no set relationship between the jaws
of the vise and the oral, aboral, left or right sides of the tissue).
The vise with its mounted tissue was transferred via a vacuum air lock to a cryochamber attached to the microscope (AMRay Biochamber; AMRay,
Bedford, MA). The tissue was viewed with a dissecting microscope while
a precooled knife fractured it at right angles to the epithelial surface. If necessary, fracturing was repeated until a clean, smooth
fracture plane perpendicular to the epithelial surface was obtained.
Some frozen, hydrated tissues were coated with gold at this point.
Others were first radiant etched for periods of up to 1 min using a
tungsten filament 10 mm from the tissue, which was heated to dull red.
Once coated with gold, tissues were mounted on a scanning electron
microscope stage cooled with a high-pressure Joule-Thompson nitrogen refrigerator (less than
180°C). The
stage was adjusted until the electron beam was parallel to the
epithelial surface. Stereo pair electron micrographs were made at a
tilt difference of 10° (5) using a lanthanum hexaboride electron source at 10 kV and an electron beam of 5 × 10
12 A (Faraday cup
measurement). Images were recorded on Polaroid 55 positive-negative 4 × 5-in. sheet film (Polaroid, Cambridge, MA). Starting near the
center of the fracture plane (total length = ~5 mm), 10 estimates of
the depth of ASL were made at intervals of 10 µm. The field of vision
was then moved 100 µm to the right along the fracture plane, and a
further 10 estimates of ASL depth were made. In total, 10 groups of 10 estimates were made. The average of all 100 estimates was taken as the
ASL depth for that specimen.
Ussing chambers were used to determine the general viability of the
tissues and the types of transepithelial ion transport present. Sheets
of tissue were mounted between lucite half-chambers with an exposed
surface area of 1.3 cm2. Warm
(37°C), oxygenated KHS was circulated across both faces of the
tissue using gas lift oxygenators. Transepithelial potential difference
(PD) was sensed by agar bridges connected via calomel half-cells to a
high-impedance electrometer. Agar bridges at the backs of the
half-chambers were connected to a voltage clamp (University of Iowa
Bioengineering, Iowa City, IA), which was used to pass current to clamp
the PD to zero. The resulting short-circuit current (Isc), which is
equal to the sum of the active ion transport processes generating the
PD (27), was displayed continuously on a chart recorder. Amiloride
(105 M, mucosal bath) was
first used to inhibit active sodium absorption. Methacholine and
forskolin (both at 10
5 M in both baths)
were then added in random order, the latter being used as a stimulator
of adenosine 3',5'-cyclic monophosphate-dependent chloride
secretion across the surface epithelium (13). Finally, chloride
secretion was inhibited with diphenylamine-2-carboxylate (2 × 10
3 M, mucosal
bath). Transepithelial electrical resistance
(Rte) was
determined every 20 s from the current needed to clamp PD from
zero to a constant value (0.5-2.0 mV) for 200 ms.
Gland secretion was measured by the "hillocks" technique (4, 6). A lucite ring held a piece of epithelium mucosal side up over the top of a lucite cylinder. The surface of the epithelium was coated with tantalum dust and viewed with a dissecting microscope. Warm, oxygenated KHS was introduced into the lucite cylinder on the serosal face of the tissue and was maintained at 37°C by a water jacket. Gland secretions were revealed as upswellings (hillocks) in the tantalum coating.
Gland openings were visualized by epi-illumination of unfixed tissue sheets, by staining of living tissue with neutral red (26), or by staining whole mounts of epithelium with alcian blue-periodic acid Schiff (PAS; see Ref. 25). Total numbers in fields of 20-70 mm2 were counted. All three methods gave similar values for the numbers of gland openings per unit area of epithelium. Goblet cells were clearly visible in the whole mounts stained with alcian blue-PAS.
The volume of glands per unit area of airway surface was obtained as
follows. Sheets of epithelium were fixed in 4% formaldehyde in saline
and embedded in paraffin. Cross sections were stained with hematoxylin
and eosin, and one end of the section was displayed at ×130
magnification on a video screen. The areas occupied by profiles of
surface epithelium and glands were traced. The slide was moved to
display the adjoining length of section on the screen, and again the
surface epithelium and gland profiles were marked. This process was
repeated until the entire length of section was scanned; sections were
~1 in. long and were covered by ~10 video screens. The length of
epithelium on each transparency was determined in inches using a
cartographic measuring wheel. The total area of gland profiles on each
transparency was determined gravimetrically in square inches. From the
known magnification on the video screen, the ratio of square inches of
gland per inch of epithelium on the transparency was converted to
square millimeter per millimeter on the section. This is the same as
the ratio of gland volume to airway surface area in microliter per
square centimeter times 102.
Data are expressed as means ± SE. Significance of differences between means was assessed by Student's paired t-test, with P < 0.05 considered statistically significant.
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RESULTS |
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As assessed from their electrical properties, tissues had good
viability on arrival at the laboratory, and this was not compromised by
dissection of the epithelium away from the underlying tissues. Figure
1, for instance, shows
Isc across two
sheets of epithelium from the anterior portion of the same trachea. In
one sheet, the underlying collagen rings had been dissected away, and,
in the other sheet, the rings were left in place. Baseline
Isc values were
similar (~150 µA/cm2) and
were inhibited ~65% by amiloride. Both tissues showed sustained increases in Isc
of ~40 µA/cm2 in response to
forskolin. Methacholine transiently stimulated Isc across the
dissected epithelium but not when the submucosal layers were intact.
Methacholine receptors are on the basolateral membrane of airway
epithelium and glands (2). Thus the failure of this agent to affect
undissected tissues may reflect the diffusional barrier provided by the
cartilage rings, which are ~0.5 cm thick. Finally,
diphenylamine-2-carboxylate markedly inhibited the
amiloride-insensitive Isc in both
tissues. Baseline
Isc and
Rte of epithelial
sheets dissected from the anterior portion of the trachea were 84 ± 33 µA/cm2 and 92 ± 30 · cm2,
respectively (means ± SD, n = 10).
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To test whether the Isc response to methacholine represented an effect on surface epithelium or glands, we compared electrical properties of epithelial sheets dissected from the anterior cartilaginous or posterior membranous portions of the trachea. The anterior portion had 1.60 ± 0.07 gland openings/mm2 (25 fields from 4 tracheas). The gland density in the posterior membranous portion was 0.82 ± 0.05 openings/mm2 (n = 22 fields from 3 tracheas). Not only were there fewer gland openings in the posterior portion, but the glands were smaller. Thus total gland volume was 17.7 ± 1.2 µl/cm2 (5 sections from 2 tracheas) in the anterior portion of the trachea and 5.3 ± 1.2 µl/cm2 in the posterior portion (4 sections from 2 tracheas). Epithelium from the anterior portion of the trachea showed maximal increases in Isc of 18.2 ± 3.6 µA/cm2 (n = 10) in response to methacholine. In about one-half of the tissues, the response to methacholine was sustained for at least 15 min. In the others, it was maximal at ~2 min and declined to baseline after ~5 min. By contrast, epithelial sheets from the posterior membranous portion of the same tracheas showed no or little response to methacholine (maximal increase = 1.7 ± 1.1 µA/cm2, n = 10). Baseline Isc, Rte, and the forskolin-induced increase in Isc (~30 µA/cm2) were not significantly different for epithelium from the anterior or posterior portions of the trachea.
Induction of gland secretion by methacholine was confirmed by the hillocks technique (Fig. 2). Within 1 min of dissection, tissues were mounted and coated with tantalum. A few small hillocks appeared shortly after coating. However, if the saline on the mucosal faces of the tissues was replaced with fresh medium at 7.5 min, this caused no further hillock formation (Fig. 2, A and B). By contrast, if methacholine was added to the serosal bath, preexisting hillocks grew in size, and new hillocks appeared (Fig. 2, C and D). The maximal number of hillocks induced by methacholine in sheets of epithelium from the anterior portion of the trachea (0.96 ± 0.03 per mm2; n = 5) was ~60% of the number of duct openings revealed by histological techniques, suggesting that not all glands responded to methacholine. Methacholine had no effect on the numbers of goblet cells or their intensity of staining with PAS-alcian blue (data not shown).
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Figure 3A shows a low-magnification electron micrograph of an unetched fracture plane revealing the characteristic pseudostratified appearance of airway epithelium. Cilia are apparent, and the mucosal surface of the cells is covered by a layer of liquid 10-15 µm in depth. Figure 3B shows a higher magnification view of a cross-fracture through another unetched tissue. Cilia are clearly visible at this magnification, and this particular specimen contained several mature goblet cells. Etching of the fracture surface made the cilia more obvious and frequently revealed two layers in the ASL, with the layer closest to the epithelium having the larger ice-crystal voids (Fig. 3C). The depth of ASL in control tissues studied in the laboratory was 23 ± 3 µm (n = 10 tracheas). This was similar to the depth of ASL in a tissue frozen at the abattoir (19 ± 3 µm).
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In two experiments, depth of ASL was determined at 1, 2, 5, 10, and 30 min of exposure to methacholine. In both experiments, the depth of ASL was greatest at 2 min and declined approximately linearly between 2 and 30 min. In further experiments, depth was measured at 2 and 30 min of exposure. Figure 4 shows three epithelial sheets illustrating the effects of methacholine on the appearance of ASL. In unstimulated tissues, frozen within 30 s of mounting on the sponge (Fig. 4A), the ASL was not much deeper than the length of the cilia (~6 µm). After 2 min of exposure to methacholine, ASL depth increased approximately sevenfold, and radiant etching revealed the presence of two layers in the ASL (Fig. 4B). After 30 min of exposure to methacholine, the depth and appearance of ASL differed little from control (Fig. 4C).
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Figure 5 shows all 100 measurements of ASL depth for two tissues from the same trachea, one untreated and the other exposed to methacholine for 2 min. The marked regional variation in depth is caused by folding of the surface epithelium. The average of all 100 estimates was taken as the ASL depth for each particular specimen. Figure 6 shows that ASL depth, so determined, was increased by a 2-min exposure to methacholine in all 10 tracheas studied. Again, in all tracheas, depth declined between 2 and 30 min of exposure (Fig. 6). When data from all 10 tracheas were combined, a 2-min exposure to methacholine increased the depth of ASL from 23 ± 6 to 78 ± 9 µm. Between 2 and 30 min of exposure, ASL depth declined to 32 ± 5 µm.
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Figure 7A shows that
amiloride had no effect on the initial increase in depth seen during
the first 2 min of exposure to methacholine but abolished the decrease
in depth between 2 and 30 min. By contrast, bumetanide
(104 M in the serosal bath)
markedly inhibited the initial increase in depth (Fig.
7B).
Untreated time-control tissues from two tracheas showed small variable changes in depth between 0 and 30 min (from 11.0 to 16.5 µm and from 14.2 to 10.9 µm). Increases in depth in response to a 2-min exposure to methacholine were normal in tissues from the same tracheas.
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DISCUSSION |
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Our studies are the first to demonstrate physiological regulation of the depth of ASL. Specifically, we show that stimulation of submucosal gland secretion causes a rapid threefold increase in depth followed by a slow decline, which is inhibited by amiloride, a blocker of active absorption of sodium across the surface epithelium.
The depth of ASL in living tissues has been measured with
microelectrodes or by light microscopy. When cultures of canine bronchial epithelium were grown with no medium added to their mucosal
surfaces ("air interface feeding"), microelectrodes revealed an
ASL depth of 15 µm (12). Rahmoune and Shephard (20) poked microelectrodes through a small window cut in the wall of guinea pig
tracheas in vivo and recorded the distance between the surface of ASL
(electrical circuit formed) and the apical membranes of the cells
(electrical interference). When the animals breathed air in which the
water vapor pressure favored condensation, the depth of ASL averaged 87 µm. Breathing dry air reduced the depth to zero in ~15 min. In
vitro, microelectrodes measured an ASL depth in isolated guinea pig
tracheas of ~200 µm when the mucosal surface was exposed to warm
humidified air. The depth decreased when dry air was blown over the
tracheal surface (19, 23). Seybold et al. (22) viewed the surface of
sheep tracheal epithelium with epi-illuminating dark-field and
bright-field optics. Using a displacement transducer with a resolution
of 0.5 µm, they determined the position of the epithelial surface
under bright-field optics and the air-liquid interface using the
dark-field mode. They found the ASL depth to be 35-50 µm. The
tracheas were exposed to
105 M acetylcholine or
epinephrine, and the depth of ASL was determined after 20 min. Next,
tissues were exposed to 10
4
M of either drug, and the depth was again measured after 20 min. Finally, tissues were exposed to
10
3 M mediator for 20 min.
Neither drug, at any concentration, altered ASL depth.
The failure of Seybold et al. (22) to detect changes in the depth of
ASL in response to autonomic agonists is surprising given that there
are a number of processes that effect liquid flows across airway
epithelium, and some of these are under autonomic control.
For instance, active absorption of sodium across human surface
epithelium in vitro removes luminal liquid at ~5
µl · cm2 · h
1
(10), a rate that would decrease the depth of ASL by ~1 µm/min. Neurohumoral agents can induce active secretion of chloride across human and dog tracheal epithelium, adding liquid to the lumen at ~3
µl · cm
2 · h
1
(10, 31). Chloride secretion across bovine tracheal epithelium can also
be stimulated by several neurohumoral agents (14). Maximal secretion
from airway glands in cats, induced by cholinergic or
-adrenergic
agents, is ~10
nl · min
1 · gland
1
(18, 26). Depending on species and airway region, gland density varies
from 1 to 10 per mm2 (1, 25). Thus
maximal gland secretion could be 60-600
µl · cm
2 · h
1
and should increase the depth of ASL by 10-100 µm/min. Finally, the marked expansion of goblet cell granules on discharge (29) could
draw liquid from beneath the epithelium and also increase ASL depth.
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We have previously established that low-temperature scanning electron microscopy of rapidly frozen tissues can be used to measure the depth of ASL (32). The advantage of this approach over other microscopic techniques is that shrinkage and some artifacts associated with processing of the tissues are avoided.
The tracheas used in these studies were kept in ice-cold saline for up to 2 h during their transfer from the slaughterhouse to the laboratory. The surface epithelium and glands were then dissected from the underlying cartilage. Ussing chamber studies, however, showed that the epithelium was in good health on arrival and was not damaged by dissecting away the underlying tissues. In fact, the values for Isc and Rte reported here for dissected epithelium are comparable to those reported for sheets of intact bovine tracheal wall by other investigators (9).
The depth of ASL in control, unstimulated tissues was 23 µm. Even though these tissues were rinsed with saline before study, the depth of their ASL was reasonably close to that seen in tissues frozen immediately on removal from the animal (19 µm).
Cholinergic agents are potent stimulators of airway gland secretion in many species (9), but their effects on bovine airway glands have not been reported. Using the hillocks technique, we demonstrated cholinergically mediated gland secretion in bovine trachea. Alcian blue-PAS staining of epithelial sheets showed no change in the numbers of goblet cells stained or in their staining intensity. This is in agreement with a previous study indicating that airway goblet cells of most mammalian species are insensitive to cholinergic stimulation (28). Finally, methacholine transiently increased Isc across epithelium from the anterior portion of the trachea but had little effect on Isc across epithelial sheets from the posterior membranous portion of the trachea, a region containing comparatively few glands.1 Thus methacholine would seem to be a specific stimulator of gland secretion, and the small transient change in Isc induced by methacholine across the anterior portion reflects the time course of methacholine-stimulated gland secretion. In most tissues, this transient was maximal at ~2 min and essentially finished within 5 min, in approximate agreement with the time course of mediator-induced gland secretion reported by others (4, 8, 18, 26).
The maximal depth of ASL occurred at approximately the same time as the
peak in gland secretion predicted from the methacholine-induced increase in Isc.
The increase in depth over the first 2 min of methacholine stimulation
(~50 µm) corresponds to a volume flow of 150 µl · cm2 · h
1.
The average density of glands in the anterior portion of bovine trachea
is ~2 mm2. Maximal gland secretion in cats is
~10
nl · min
1 · gland
1 (18, 26). With the
assumption of similar flow rates in bovine airway glands, maximal
secretion should be 120 µl · cm
2 · h
1,
in good agreement with the volume flow calculated from the change in
depth. The liquid component of gland secretions is probably produced
secondarily to active sodium-linked secretion of chloride (1, 33).
Consistent with this, we found that the initial increase in depth was
inhibited by bumetanide, a blocker of epithelial chloride secretion.
Between 2 and 30 min of methacholine exposure, ASL showed a decline in
depth of ~40 µm. This decline was blocked by amiloride, thereby
directly implicating osmotic gradients created by active absorption of
sodium as the mechanism behind the depth decrease. Further support for
this idea is the good quantitative agreement between transepithelial
volume flows measured directly or estimated from the decline in ASL
depth. Thus baseline liquid absorption across primary cultures of
bovine tracheal epithelium is ~5
µl · cm2 · h
1
and is abolished by amiloride (S. Uyekubo, J. H. Widdicombe, and S. S. Miller, unpublished observation). The decline in depth from 75 to 30 µm between 2 and 30 min of methacholine exposure yields a similar
value for liquid absorption across native tracheal epithelium of ~9
µl · cm
2 · h
1.
Our results contrast with those of Seybold et al. (22), who failed to see an effect of acetylcholine on the depth of ASL in isolated sheep tracheas. However, their measurements of depth were made after a 20-min exposure to drug. Our results in the bovine trachea suggest that gland secretion is transient and is followed by liquid reabsorption. Therefore, it is possible that Seybold et al. missed an initial transient increase in depth. We also note that the baseline depth of ASL was somewhat deeper (~50 µm) in the studies of Seybold et al. than in ours (~25 µm).
In both control and methacholine-stimulated tissues, radiant etching frequently revealed the presence of two layers. The layer closest to the epithelium had large ice-crystal voids, and we speculate that it corresponds to the periciliary sol. The upper layer with smaller voids may be the mucous gel. In control tissues, the depth of the putative sol was approximately the same depth as the length of the cilia (Figs. 3A and 4A), as would be expected for optimal mucus transport.2 Further experimental approaches are needed, however, to determine whether the regions of small and large ice- crystal voids do indeed correspond to the gel and sol.
In conclusion, our results establish that gland secretions can cause large and rapid increases in the depth of ASL in vitro, followed by a return to baseline effected by active absorption of sodium across the surface epithelium.
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ACKNOWLEDGEMENTS |
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We thank Walt Finkbeiner (Department of Pathology, University of California, San Francisco) for help with the histological analysis of gland volumes and Rachel Kline and Vy Crawford for help with manuscript preparation.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-42368 and HL-52161, Cystic Fibrosis Foundation Grant G772, and Tobacco-Related Disease Research Program Grant 4RT 0382.
1 The ratio of gland volume in the posterior portion to gland volume in the anterior portion was 0.30 ± 0.07. The corresponding ratio of Isc responses to methacholine was 0.09 ± 0.06, a significantly lower value. Two possible reasons for this difference are that the relation between gland secretion and change in Isc is not linear and/or glands in the posterior membranous portion are less sensitive to methacholine. A third possibility is that the posterior epithelial sheets used in Ussing chambers were more stretched than those used for histology; stretch will reduce the ratio of gland volume to epithelial surface area. The important point, however, is that the larger volume of glands in the anterior portion is associated with a larger Isc response to methacholine.
2 Although the changes were not quantified, the depths of both the putative sol and gel increased after stimulation with methacholine (Fig. 4B). Thus, during stimulation, the putative sol became considerably deeper than the length of the cilia, suggesting that mucocilary clearance should be impaired at the height of gland secretion.
Address for reprint requests: J. H. Widdicombe, Children's Hospital Oakland Research Institute, 747 52nd St., Oakland, CA 94609.
Received 3 September 1997; accepted in final form 17 December 1997.
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