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1 Cystic Fibrosis Research Laboratory, Stanford University, Stanford 94305-2130; and 2 Ethicon Endo-Surgery, Incorporated, Stanford, California 94305
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We describe an optical method to quantify
single- gland secretion. Isolated tracheal mucosa were mounted at the
air-Krebs interface and coated with oil. Gland secretions formed
spherical bubbles that were digitally imaged at intervals, allowing
rates of secretion to be calculated. We monitored 340 glands in 54 experiments with 12 sheep. Glands secreted basally at low rates
(0.57 ± 0.04 nl · min
1 · gland1, 123 glands) in tissues up to 9 h postharvest and at lower rates for up
to 3 days. Carbachol (10 µM) stimulated secretion with an early
transient and a sustained or oscillating phase. Peak secretion was
15.7 ± 1.2 nl · min1 · gland1 (60 glands); sustained secretion was 4.5 ± 0.5 nl · min1 · gland1 (10 glands). Isoproterenol and phenylephrine (10 µM each) stimulated only
small, transient responses. We confirmed that cats have a large
secretory response to phenylephrine (11.6 ± 3.7 nl · min1 · gland1, 12 glands), but pigs, sheep, and humans all have small responses (<2
nl · min1 · gland1).
Carbachol-stimulated peak secretion was inhibited 56% by bumetanide, 67% by HCO
carbachol; phenylephrine; cystic fibrosis transmembrane conductance regulator; lung disease; mucociliary clearance
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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SUBMUCOSAL GLANDS
are a major source of the mucus that coats the luminal surface of
cartilaginous airways. Submucosal glands are complex structures
comprising multiple tubules that feed into a large collecting duct that
narrows on its way to the airway surface (25). The tubules
are lined with mucous cells and serous cells. Serous cells are
predominant in the acini so that their watery secretions wash over
mucous cells and then mix with mucins in the collecting duct before
being expelled (24). Serous cell secretions are rich in
antimicrobials and antioxidants that are important components of
mucosal defense (3). In the genetic disease cystic
fibrosis, gland malfunction may contribute to the genesis of airway
infections. Cystic fibrosis transmembrane conductance regulator (CFTR),
the protein that is defective in cystic fibrosis, is heavily expressed
in gland serous cells (9). Cholinergic stimulation induces
gland secretion in porcine bronchi that is driven by Cl
and HCO
As a prelude to comparing submucosal gland function in normal and cystic fibrosis airways, we are developing methods to assess the function of individual glands. Functional data can then be combined with gland morphology obtained by other methods (26). Structural and functional studies need to be combined because glands become larger and more numerous in response to airway disease, and these changes must be considered when comparing secretion rates. In addition, individual glands vary in both size and cellular components (31) and may be differentially affected by disease. In prior studies of secretion rates of single glands, mucus was sampled with constant-bore micropipettes. These were either applied directly to the gland duct orifice (6, 10, 11, 22, 39) or used to collect bubbles of mucus that had formed under an oil coating (29). These methods are accurate but tedious and thus limit both the number of glands sampled and the minimal sampling interval. To allow rapid, frequent interval assessment of secretion rates in multiple, individually localized glands, we have modified the methods of Quinton (29) so that we can measure secretion rates optically with a digital video camera.
In this study, we applied these methods to tracheal submucosal glands of sheep. No adequate animal model of cystic fibrosis lung disease is presently available because CFTR-deficient mice do not develop airway disease. Fortunately, continuing improvements in cloning methods presage the development of other CFTR-deficient animals, some of which have airways more similar to humans. Sheep have particular advantages in this regard because they are suitable for cloning (4) and because prior studies of sheep airways indicate similarities with human airways (36), including a good complement of submucosal glands (5, 23).
However, there have been no prior functional studies of intact
submucosal glands in sheep. In this study, we used our newly developed
optical methods to quantify gland secretion in sheep. We discovered a
marked species difference in response to 
-adrenergic stimulation,
document an extreme range of secretory rates across individual
submucosal glands, and show that gland secretion in sheep, as in pigs
(2, 16, 37), depends on both Cl and
HCO
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animal tracheas were harvested <1 h postmortem from 16 wethers sheep (Suffolk-Rambouillet), 4 female pigs (Yorkshire), and 2 male cats, all adult. All animals had been killed with pentobarbital sodium injection after acute experiments unrelated to the present studies. Two pieces of human trachea were obtained as surgical trimmings from lung transplant donors. All tracheas were maintained until used in ice-cold Krebs-Ringer bicarbonate buffer (KRB) bubbled with 95% O2-5% CO2. The KRB composition was (in mM) 115 NaCl, 2.4 K2HPO4, 0.4 KH2PO4, 25 NaHCO3, 1.2 MgCl2, 1.2 CaCl2, and 10 glucose (pH 7.4). Osmolarity was measured on a Westcor vapor pressure osmometer and was adjusted to ~290 mosmol/l. To minimize tissue exposure to endogenously generated prostaglandins during tissue preparation and mounting, 1.0 µM indomethacin was present in the bath throughout the experiment unless otherwise indicated.
For each experiment, a tracheal ring of ~1.5 cm was cut off, opened up along the dorsal (posterior) fold in ice-cold, oxygenated KRB, and pinned mucosal side up on a pliable silicone surface. Only the cartilaginous portion of trachea was used. The mucosa with underlying glands was carefully dissected from the cartilage and connective tissues and mounted in a 35-mm, Sylgard-lined plastic petri dish with the serosa in the bath (2-ml volume) and the mucosa in air. The dish was transferred to a temperature- and humidity-controlled chamber (Medical Systems, Greenvale, NY) and was gradually warmed to room temperature. The tissue surface was blotted dry and then further dried with a gentle stream of inert gas, after which 30-40 µl of water-saturated mineral oil were placed on the surface. The tissue was then warmed to 37°C at a rate of ~1.5°C/min. Some secreting glands were observed at room temperature, and many more started secreting as the bath was warmed. The process of cleaning, drying, and oiling the epithelium did not appear to stimulate or inhibit secretion because similar secretion was observed in tissues or areas not so treated. "Basal" secretion refers to the secretion observed in otherwise unstimulated tissues.
Optical measures.
The experimental setup is shown in Fig.
1A. For most experiments, the
chamber was continuously superfused with warmed, humidified 95%
O2-5% CO2 to minimize evaporation and to
maintain bath pH at 7.4. The preparation was obliquely illuminated with
a fiber- optic illuminator. The appearance of the secreted mucus
droplets was strongly dependent on details of illumination that were
adjusted empirically for each preparation. A digital video camera
(Logitech) was mounted on one optical tube of a dissecting microscope.
The image projected on the charge-coupled device sensor was captured as
a bitmap representing an area ~6.25 mm2. The resolution
was 640 × 480 pixels, yielding ~49,000 pixels/mm2.
Digital images were captured at intervals of 1-5 min using
software supplied with the camera and were stored on disk for
subsequent analysis using a modification of National Institutes of
Health Image software (version 4.2; Scion, Frederick, MD). The captured image was calibrated using a 0.5-mm grid. The area from the perspective of the optical axis of the microscope of each droplet was then measured
and converted to volume (V) using the spherical approximation V = 4/3
r3, where r is the
radius. During analysis, we discovered that measurements in the
leftmost 35 pixels of the image field were underestimated by the
software package if measured with the freehand or circle tools. All
other areas were accurate, as were measurements in this area made with
rectangle or line measurement tools. If an image in the leftmost field
was outlined with freehand or circular tools and the image was then
moved out of the area, the measurement was again accurate.
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10%, both because of the distribution of contact angles and because many contact angles are underestimated because of the uneven surface of
the epithelium. Many ducts exit in shallow pits, so that when viewed
from the side, fully spherical droplets appear to have a contact angle
<180°. However, although these arguments suggest that our average
rates are accurate, individual rates for single glands will be strongly
affected by these factors (see below).
To further validate our spherical approximations of bubble volumes and
to ensure that no fluid was partitioning from the small droplets in the
oil, control experiments were carried out in which bubbles of water or
buffer with different known volumes was injected in oil using
silanized, constant-bore microcapillaries (Drummond Scientific) and
then followed over time under normal experimental conditions. The
difference between the volume of a fluid bubble measured in a
microcapillary and the digitally calculated spherical values was within
±10% (n = 11). Side visualization of the injected bubbles with a prism revealed no significant vertical distortion. The
x- and y-axes were within ±3%, which is within
our measurement error. Volumes were observed over a period of 3 h
in bubbles of buffer (Fig. 1D) or water (data not shown).
The smallest bubble of buffer (~33 nl) retained its original volume
for at least 5.5 h (data not shown). As an additional control for
possible optical distortion of images caused by the oil coating, a
0.5-mm calibration grid was covered with oil, and the images obtained
were compared with the same grid in air. No size differences were
observed between the two conditions.
For HCOReagents. All compounds were obtained from Sigma unless otherwise indicated and were maintained as stock concentrations. Carbachol, phenylephrine, isoproterenol, phentolamine, and propranolol were dissolved in deionized water at a stock concentration of 10 mM. Other stock solutions were as follows: indomethacin, 10 mM in ethanol; acetazolamide, 1 M, and atropine, 10 mM, in DMSO; bumetanide, 0.1 M in an alkaline solution; and TTX, 0.1 mM in 0.2% acetic acid.
Statistics. Data are means ± SE. Student's t-test for paired or unpaired data or the Mann-Whitney U-test was used as appropriate to compare the means of different treatment groups. The difference between the two means was considered to be significant at P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Our results are based on sampling 340 single glands in 54 experiments from 12 sheep, with most data obtained from 5 sheep. We monitored 6.3 ± 0.3 glands (range 2-13)/experiment.
Basal secretion.
Basal secretion (Fig. 2,
A and B) was quantified 1-9 h postharvest
for 123 glands from 7 sheep (Fig. 3). No
differences in average secretion rates were observed over this 9-h time
period. In addition, there was no consistent increase or decrease in
basal secretion rates as a function of time in the experimental setup. The mean basal secretion rate for all 123 glands averaged over a 20-min
period was 0.57 ± 0.04 nl · min1 · gland1.
Variation in secretion rates among individual glands constituted the
largest source of variation in our experiments. Extreme variation could
occur within a single tissue preparation. For example, within a
contiguous 6.25-mm2 area of tracheal tissue from a single
sheep exposed to identical treatment, the fastest gland secretion rate
was 25 times the slowest rate, i.e., 0.08 and 1.98 nl · min1 · gland1 in a
1.5-h-old tissue preparation containing 9 glands (Fig. 3). Such
intergland differences greatly exceeded differences in average basal
secretion rates among sheep, which varied only fourfold, i.e., from a
minimum of 0.23 ± 0.08 to a maximum of 0.92 ± 0.21 nl · min1 · gland1.
However, a portion of this wide range of variation may be an artifact
of the measurement method. If a larger droplet from a rapidly secreting
gland wetted the surface so that its contact angle went from 180 to
135°, its apparent volume would then increase by 11%; if the contact
angle was 90°, its apparent rate of increase would be two times its
actual rate.
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-adrenergic (10 µM phentolamine), or
-adrenergic (10 µM propranolol) stimulation, alone or in
combination, also did not diminish basal secretion (data not
shown). These treatments eliminate several potential types of
stimulated secretion but do not rule out the possibility that what we
term basal secretion is actually a response to an unidentified stimulus.
The mean basal secretion rate in each preparation declined over time
postharvest. Compared with tissues tested from 1 to 9 h postharvest,
basal secretion rates were reduced to ~44% in tissues tested
17-30 h postharvest (0.25 ± 0.03 nl · min1 · gland1, 168 glands in 8 sheep, P < 0.01, Mann-Whitney) and fell to
11% in tissues tested 42-56 h postharvest (0.06 ± 0.03 nl · min1 · gland1, 49 glands in 3 sheep, P < 0.01 vs. 17-30 h,
Mann-Whitney; Fig. 4). These values
considerably underestimate the magnitude of the decline in the basal
secretory potential of the tissues because they only provide rates for
active glands and do not indicate the proportion of glands that had
become inactive or had basal secretion rates too low to measure.
Cursory observations indicate that the proportion of basally secreting
glands also declined over the same time period. We did not quantify
that decline because our present method was optimized to provide
accurate secretion rates for individual glands by sampling a small area
of tissue. This sampling was not done randomly but instead focused on
areas of actively secreting glands (see DISCUSSION).
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Secretion stimulated by carbachol.
Gland secretion was markedly increased by the cholinergic agonist
carbachol (10 µM; Fig. 2, C and D). The
response to carbachol included a short-latency, transient peak followed
by sustained secretion that was about one-third of the peak response
(Fig. 5). Mean peak secretion rates to
carbachol were 15.7 ± 1.2 nl · min1 · gland1 when
measured at 1-min intervals (60 glands in 5 sheep), with peak responses
in some glands reaching ~38
nl · min1 · gland1.
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1 · gland1 (Fig.
5). As with basal secretion, rates of carbachol-stimulated secretion
varied >10-fold among glands, i.e., from 3.1 to 33.4 nl · min1 · gland1 in one
tissue preparation containing seven glands in an area of 6.25 mm2. In addition, when secretion rates were tracked at
1-min intervals, we observed marked differences among glands in the
temporal patterns of secretion, including oscillations in the secretory
rate (Fig. 6). In spite of these large
gland-to-gland variations, the mean peak secretory responses to
carbachol across sheep varied only 1.5-fold (Fig.
7). In contrast to the decline in basal
secretion, the rate of gland secretion stimulated by carbachol was
stable for a period of at least 1 day after harvesting (Fig.
8).
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Secretion stimulated by phenylephrine.
The -adrenergic agonist phenylephrine (10 µM) stimulated peak
gland secretion of only 0.8 ± 0.1 nl · min1 · gland1 (39 glands, 5 sheep), a value ~5% of the average peak response to
carbachol (Fig. 9A,
inset). The response to phenylephrine was transient, with
secretion rates returning to basal values within 5-10 min after
stimulation. Thus, when compared over longer time periods, the response
to -adrenergic stimulation is trivial compared with cholinergic
stimulation.
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-adrenergic agonists (22, 29). To determine if the difference represented a
species or methodological difference, we also studied secretion in
tracheas from two cats, four pigs, and two humans. In agreement with
the prior reports, we observed that phenylephrine stimulated copious gland secretion in the cat of a magnitude similar to the response to
carbachol (Table 1). In contrast, pigs
and humans responded, like sheep, with large responses to carbachol but
with small, transient responses to phenylephrine (Fig. 9B,
inset; Fig. 9C; and Table 1).
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Secretion stimulated by isoproterenol.
The -adrenergic agonist isoproterenol (10 µM) stimulated peak
gland secretion 1.8 ± 0.7 nl · min1 · gland1 (18 glands, 2 sheep), equivalent to ~9% of the average peak response to
carbachol. The response to isoproterenol was transient and returned to
baseline within 10~20 min after the treatment (Fig. 10).
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Inhibition of secretion with bumetanide.
In many epithelia, fluid secretion depends on secretion of
Cl or HCO cotransporter NKCC1 is
a common means for elevating intracellular Cl
concentration, and in many tissues, its inhibition with bumetanide eliminates the major portion of secretion. We found that 100 µM bumetanide had highly variable effects on basal gland secretion, reducing secretion of individual glands by 4-83%. The mean
residual basal secretion after bumetanide was 65 ± 19% (25 glands in 3 sheep, P = 0.06, not significant; Fig.
11A). In contrast, for
carbachol-stimulated secretion, the inhibitory effect was more
effective and consistent, reducing peak carbachol-stimulated secretion
to 45 ± 5% of the control value (24 glands from 3 sheep,
P < 0.01, Fig. 11B).
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Inhibition of secretion by HCO
Inhibition of secretion by bumetanide plus
HCO and HCO
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Advantages of the single gland optical method. When studying differences in the amount or composition of airway secretions caused by different agonists, species, or region of airway or disease state, single-gland studies can distinguish factors that are confounded in pooled samples. Such factors include differential recruitment or loss of distinct populations of glands, changes in gland number or gland size, or temporal secretion properties.
Prior studies of secretion rates by individual airway glands are relatively rare (Table 1). In an early study of individual gland secretions, powdered tantalum was placed on the airway surface to reveal hillocks of mucus that formed above gland ducts that were subsequently visualized with neutral red staining (27). This method is typically used to count secreting glands (41), but, by making assumptions about the shape of the hillocks, it can also be used to quantify secretion for individual glands (7, 13-15, 28). For more accurate quantification of single gland secretion, Quinton (29) developed the oil-coating method and then collected secretions from excised tissues at timed intervals with constant-bore micropipettes. Ueki and colleagues (39) developed a similar micropipette method for collecting secretions from individual glands in situ without using oil. They measured single-gland secretion induced by autonomic stimulation (39), mechanical stimulation of the larynx (11), gastric irritation (10), and various autonomic mediators (22). In more recent studies, single-gland secretion in pig bronchi was studied with video microscopy with a water-immersion lens (18). Secretion rates were not quantified, but the latency of the rapid response to carbachol was estimated by dilation of the gland duct and emergence of particles from the gland. Finally, methods to study isolated glands have been developed (30, 34) and used to quantify mucus secretion in the cat with a variety of markers, such as glycoconjugates and Na+ efflux (see Ref. 33 and references therein). The discontinuation of the micropipette method is understandable, given the technical difficulties involved. Its restriction to cats probably arises from the relative ease with which cat mucus can be collected. In our studies, we find cat mucus to be less viscous than the mucus of the other species studied (unpublished data). The collection and manipulation of tiny quantities of airway mucus from other species presents technical challenges that are circumvented by optical methods. Compared with micropipette collections, the optical method allows secretion rates to be quantified more frequently and in more glands. The average gland number per experiment was only six glands in the present work because we sampled a small area (6.25 mm2) to allow greater resolution. Rapid increases in the cost effectiveness of digital imaging will make this trade-off unnecessary. The relative accuracy of pipette and optical methods was not specifically compared, as each would appear to depend mainly on details of execution. Therefore, it is interesting that they yield very similar estimates of rates (Table 1) even before applying any corrections to our data for surface wetting (see METHODS). This could be a fortuitous result of offsetting errors. In the micropipette method, small gland openings and slowly secreting glands are probably underrepresented in the sample. In the optical method, these slowly secreting glands are included, whereas some of the fastest secreting glands are excluded because of merging, which would tend to reduce the average rate, but the volume of droplets that partially adhere to the surface is slightly overestimated. We are further developing our method to decrease this source of error. In micropipette studies, the composition and physical properties of single-gland mucus can be studied using microanalytical techniques (22, 29). These can be highly accurate, but they require great skill and are labor intensive. The optical methods we have developed can easily be adapted to measure physical and chemical properties of individual gland secretions. In experiments now underway in collaboration with Drs. Alan Verkman and S. Jayaraman, Na+, Cl, and pH are quantified in situ by
injecting ratiometric fluorescent indicators in the undisturbed bubbles
of mucus as they are secreted under oil from the gland duct. Viscosity
is measured using fluorescence recovery after photobleaching.
Limitations of the method. This method is not optimal for long-term monitoring of secretion because the accumulating mucous bubbles fuse or lose their spherical shape. We achieve long-term monitoring by periodically collecting the secretions, but the collections introduce gaps in the monitoring and are labor intensive. Thus this method complements the method of Ballard et al. (2) and Trout et al. (37, 38) in which bulk mucus is collected for several hours from the entire bronchi. The optical method also eliminates the natural interaction between gland secretions and the surface epithelium. For our present purposes, that simplification is useful, but it is also important to determine how the transport properties of surface epithelium and glands interact to determine the depth and composition of airway surface liquid, which in turn affects mucociliary clearance (40, 41).
Potential artifacts of the optical method arise mainly from the treatment required to obtain good optical images. Optimal imaging requires a flat, dry surface, and this requires the mucosa to be dissected free of cartilage, stretched, cleaned, dried, and oiled. These treatments might influence gland secretion, although no obvious differences were observed when comparing gland secretions from tracheal samples that had intact cartilage and were only lightly blotted. However, it is possible that surface drying and oil coating might block some gland duct openings and prevent or slow secretion unless strongly stimulated. That could contribute to the wide variation observed in basal secretion, including the lack of such secretion in some glands that did respond to stimulation.Basal secretion.
Basal gland secretion in situ is >10-fold greater than secretion of
isolated mucosa (Table 1), indicating the importance of parasympathetic
tone. About one-half of the basal secretion in isolated mucosa was
resistant to combined treatment with bumetanide and
HCO
Evidence for subpopulations of glands.
In prior studies of individual gland secretion, a wide variation in
secretion rates was noted (29, 39) and that was again observed in our studies. However, whereas gland secretion rates in
single cats were reported to vary 2- to 3-fold
(39), we documented >12-fold differences in
phenylephrine-stimulated secretion, i.e., 2.9 and 36.6 nl · min1 · gland1 within
one small patch of a cat tracheal preparation containing 10 glands.
Basal and carbachol-stimulated gland secretion in sheep trachea showed
similar wide variations in secretion rates by individual glands.
Moreover, gland secretion rates did not form a normal distribution,
suggesting that discrete gland populations exist. Previous studies
noted three different types of gland morphology (18) and
detected marked differences among glands in the expression of the
glycoprotein gene MUC7 (31). It will be
important to determine if any of these features are correlated, if
glands show diversity in other features, and if any of these features
have functional consequences. If distinct subpopulations of glands can
be identified, it is possible that they will be differentially affected
by airway diseases.
Complex responses to carbachol. On average, carbachol produced a transient peak in gland secretion that was ~28-fold greater than basal secretion (<9-h-old tissue preparations), followed by sustained secretion of approximately one-third of the peak rate, with the same wide variation in actual rates observed for basal secretion. When examined individually, different glands showed distinct temporal response patterns, with prominent oscillations of rate in some glands (Fig. 6D). Variations in rate might arise from several sources. They could have a trivial basis. For instance, Inglis et al. (18) observed a transient block of secretion by a particle that occluded a duct. They could be oscillations in myoepithelial cell tension, although none were reported in direct measures of tension of isolated cat and dog glands (34). Finally, the oscillations, which had periods of 2-3 min, might reflect oscillations of fluid secretion secondary to oscillations of intracellular Ca2+ concentration. Oscillations with similar frequency occur in Calu-3 cell monolayers when stimulated with isoproterenol or thapsigargin (26, 32).
There is disagreement in the literature about whether responses to carbachol are transient or sustained. In cat trachea (29) and pig distal bronchi (37), mucus secretion was sustained (for at least 2 h in pig), whereas, in bovine trachea, secretion returned to baseline within ~5 min (41). It is not yet known if these are species or methodological differences. Responses to carbachol in the sheep were robust. We observed undiminished responses to carbachol in glands that had been isolated for >48 h previously. This is consistent with evidence that contractions of tracheal submucosal glands to cholinergic agonists were undiminished up to 3 days postharvest (34).Ineffectiveness of adrenergic stimulation.
Neither - nor -adrenergic stimulation was an effective agonist
for mucus secretion in sheep. A small response to the -adrenergic agonist isoproterenol is consistent with prior reports for cats (22, 29). In contrast, the ineffectiveness of the
-adrenergic agonist phenylephrine was unexpected because in cats
phenylephrine is similar in effectiveness to cholinergic stimulation
(29, 39). We confirmed a large response to phenylephrine
in cats but went on to show that it is ineffective in sheep, pigs, and humans. The basis for these species differences is presently unknown.
Role of NKCC-mediated (bumetanide-inhibitable)
Cl secretion.
The inhibitory effect of bumetanide on gland mucus secretion has been
studied in four species with very different results. Bumetanide
inhibition of cholinergically stimulated secretion was 55% in sheep
(this paper), 70% in pigs (37), and 85% in cows
(41). In contrast, phenylephrine-stimulated gland
secretion in cats was unaffected by bumetanide (6).
Corrales et al. (6) interpreted the insensitivity to
bumetanide (and to anion substitutions) to mean that gland fluid
secretion was passively produced after the release of osmotically
active components of secretory granules. However,
phenylephrine-mediated secretions in cats are less viscous than
cholinergically mediated secretions (22), and, in pigs, inhibition of fluid secretion with bumetanide and dimethylamiloride caused scantier, thickened secretions (38). On the basis
of these results, and additional arguments that follow (see below), we
suggest that gland secretion may rely on an as yet unspecified way on
HCO
Role of HCO
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ACKNOWLEDGEMENTS |
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We thank Jennifer Arends (Veterans Affairs Hospital at Palo Alto, CA) who made sheep tracheas available for our research and who aided in tissue harvesting, Dr. Keith J. Jenné, University of California, Berkeley, who provided 2 cat tracheas, and Drs. Bruce Reitz and Gerald Berry, Stanford University Hospital, who provided scrap tracheal trimmings from lung transplant donors. Dr. Jonathan Widdicombe provided detailed criticism of an earlier draft of the manuscript, and Valerie Baldwin provided technical assistance.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants DK-51817 and HL-60288 and by the Cystic Fibrosis Foundation.
Address for reprint requests and other correspondence: J. J. Wine, Cystic Fibrosis Research Laboratory, Bldg. 420 (Jordan Hall), Stanford Univ., Stanford, CA 94305-2130 (E-mail: wine{at}stanford.edu).
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 February 2001; accepted in final form 22 March 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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