Optical method for quantifying rates of mucus secretion from
single submucosal glands
Nam Soo
Joo1,
Jin V.
Wu1,
Mauri E.
Krouse1,
Yamil
Saenz2, and
Jeffrey J.
Wine1
1 Cystic Fibrosis Research Laboratory, Stanford University,
Stanford 94305-2130; and 2 Ethicon Endo-Surgery,
Incorporated, Stanford, California 94305
 |
ABSTRACT |
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 · gland
1, 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 · min
1 · gland
1 (60 glands); sustained secretion was 4.5 ± 0.5 nl · min
1 · gland
1 (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 · min
1 · gland
1, 12 glands), but pigs, sheep, and humans all have small responses (<2
nl · min
1 · gland
1).
Carbachol-stimulated peak secretion was inhibited 56% by bumetanide, 67% by HCO
replacement with HEPES, and 92% by
both. The distribution of secretion rates was nonnormal, suggesting the
existence of subpopulations of glands.
carbachol; phenylephrine; cystic fibrosis transmembrane conductance
regulator; lung disease; mucociliary clearance
 |
INTRODUCTION |
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
(1, 17, 18, 37) and involves
CFTR (2). If serous cells depend on CFTR for fluid secretion, as indicated by studies of primary cultures of gland cells
(19, 42) and cell line models of serous cells (12, 26, 32), the resulting alterations in gland secretion might compromise mucosal defenses.
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
transport.
 |
METHODS |
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.

View larger version (71K):
[in this window]
[in a new window]
|
Fig. 1.
A: experimental setup. KRB, Krebs-Ringer bicarbonate
buffer. B: schematic diagram showing formation of a
spherical droplet (4/3 r3) of mucus at the
mouth of a single-gland duct under oil. Arrows denote direction of
liquid secretion. C: representative top and side views of
mucus bubbles. Because of difficulties in placing the prism, we did not
attempt to image the identical field. The two images are from adjacent
areas in the same tissue, taken within 5 min of each other.
D: control experiments showing stable volume over time for
droplets of KRB in oil. Images are top views of ~285- and ~75-nl
droplets at start (a) and end (b) of 3-h
incubation. Scale bar, 0.5 mm.
|
|
When the surface was properly cleaned and dried, the volume of secreted
bubbles was easily determined because they usually formed spheres that
did not contact the surface except for continuity of the mucus at the
gland opening. Nonspherical bubbles could usually be approximated by a
sphere. The error of this approximation was proportional to the square
root of the short axis/long axis (because this ratio is always <1, the
rate calculation may underestimate the actual secretion rate). In some
cases, the droplets obviously adhered to the surface and spread, and
these were omitted from the analyses. With continued secretion, spheres
from adjacent duct openings could merge, and in such cases, measurement
was discontinued. A schematic diagram showing a mucous bubble forming above a gland is shown in Fig. 1B.
To assess the possibility that mucus bubbles that appeared to be
spheres might actually be partially adherent, we visualized them from
the side using a prism (Melles Griot, Irvine, CA).
Control experiments were done in pigs because sheep were no longer
available. This revealed that, although the bubbles were clearly
spherical (i.e., there was no flattening or elongation in the
z-axis), many appeared to have contact angles <180° (Fig. 1C). To estimate the proportion of droplets with different
contact angles, two observers independently rated 175 and 82 droplets, respectively, assigning them to one of five categories. The percentages falling into each category for the two raters were as follows: for
contact angle 180° = 81 and 30%; 135° = 14 and 39%; 90° = 4 and
22%; 45° = 0 and 7%; <45° = 0 and 1%. The agreement between the
two raters was poor when deciding whether a droplet was closer to
180° or 135° contact angle, but the raters put 96 and 69% of the
droplets into the combined category. Also, the droplets rated as having
contact angles <45° can be discounted because these can be
distinguished by visual cues when viewed from above and are not
included in our optical measures. Eliminating those droplets gives the
following: >135° = 96 and 76%; 90-135° = 4 and 24%. Using
the worst estimates from rater 2, and making the worst case assignment that 76% of droplets have contact angles = 135° and 24% have contact angles = 90°, our secretion rates should be
reduced by a maximum of 21%. This follows because the volume of a
droplet with a contact angle of 135° is only 6% less than the volume
of a full sphere. A more reasonable correction factor is close to
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 HCO
-free experiments, all HCO
in the Krebs buffer was replaced either with 25 mM HEPES or 1 mM HEPES
plus 24 mM NaCl that had been pregassed with humidified 100%
O2. The 1 and 25 mM levels of HEPES both maintained the
bath pH at 7.4 after gassing with O2. The 25 mM HEPES
solution provided the best control for intracellular pH, whereas the 1 mM HEPES solution minimized the chance that alterations in secretion
were secondary to ion gradients established between the bath and gland lumen.
Water-saturated mineral oil was prepared by sonicating (~5 min) a
mixture of mineral oil and water (50:50 by vol) and was stored at
4°C. Before each experiment, water-saturated oil was vortexed and
briefly centrifuged at room temperature. All pharmacological agents
were diluted to a final concentration with prewarmed, appropriately gassed bath solution immediately before addition to the serosal bath
via complete bath replacement. For washout, the bath was replaced
completely at least three times. During periods without stimulation,
the bath was replaced at 10- to 15-min intervals with fresh, 37°C KRB
solution that had been constantly gassed with 95% O2-5%
CO2. No changes in secretion rate were associated with bath changes.
Reagents.
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.
 |
RESULTS |
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 · min
1 · gland
1.
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 · min
1 · gland
1 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 · min
1 · gland
1.
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.

View larger version (122K):
[in this window]
[in a new window]
|
Fig. 2.
Representative images of basal and carbachol-stimulated gland
secretion. A and B: basal secretion 5-10 min
after applying oil to the epithelium (A) and 20 min later
(B). C and D: carbachol-stimulated
secretion immediately before (C) and 2 min after
(D) carbachol. t, Time (min).
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3.
Single-gland basal secretion rate vs. time postharvest.
Each symbol plots the rate of basal secretion for a single gland
averaged over a 20-min period beginning at the indicated time
postharvest. Data for 123 glands from 7 sheep (S2-S16).
|
|
We considered it possible that at least some component of basal
secretion might be a response to some combination of trauma from the
dissection, mechanical stimulation from the tissue preparation, or
temperature fluctuations. However, basal secretion was not inhibited by
1 µM indomethacin or 0.1 µM TTX. Antagonists of cholinergic (10 µM atropine),
-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 · min
1 · gland
1, 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 · min
1 · gland
1, 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).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Decline in basal secretion over days postharvest. Each
symbol represents the average basal secretion rate over a 20-min period
for 2-13 glands from a single experiment. The mean number of
secreting glands per experiment did not change over this period (6.1 glands/experiment for the period 1.5-8.5 h and for 42-56 h),
but this is at least partly attributable to the experimental protocol,
which scanned the tissue to find areas of secreting glands.
|
|
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 · min
1 · gland
1 when
measured at 1-min intervals (60 glands in 5 sheep), with peak responses
in some glands reaching ~38
nl · min
1 · gland
1.
In pig trachea, ACh produces sustained mucus secretion for at least
2 h (37). To establish a basis for correlating
secretion of individual glands with sustained secretion of the entire
epithelium, we used the average value of sustained secretion after the
initial transient. For 10 glands in 5 sheep, sustained secretion to
carbachol, defined as the period 5-20 min poststimulation, was
4.5 ± 0.5 nl · min
1 · gland
1 (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 · min
1 · gland
1 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).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 6.
Variations in secretory responses of single glands to carbachol.
Each graph shows the response rates of an individual gland. Responses
were selected to show large transients (A and B),
small transient (C), and oscillating response
(D). Carbachol (10 µM) was present from 20 to 40 min.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 7.
Mean peak secretory responses to carbachol are similar
across sheep. Each bar is average of all glands tested from one sheep.
Smallest and largest mean responses differ ~1.5-fold. No. of glands
per sheep is shown above bars.
|
|

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 8.
Single-gland peak secretion rates to carbachol as a
function of time postharvest. Each symbol represents the peak secretion
rate of a single gland sampled at 1-min intervals after stimulation
with 10 µM carbachol. Data are from 60 glands in 5 sheep
(S12-S16); all glands from one sheep have the same symbol. Time
scale is compressed after 5-h time point. Note scale break on
x-axis.
|
|
Secretion stimulated by phenylephrine.
The
-adrenergic agonist phenylephrine (10 µM) stimulated peak
gland secretion of only 0.8 ± 0.1 nl · min
1 · gland
1 (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.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 9.
Phenylephrine-stimulated gland secretion. A:
representative responses to 10 µM phenylephrine and 10 µM carbachol
(Carb, inset) in glands from one sheep. Each closed circle
denotes average basal and stimulated secretion rates of 3-8
glands. B: representative secretion rates of phenylephrine-
and carbachol (inset)-stimulated gland secretions from a
cat. Each closed square represents average basal and stimulated
secretion rates of 8~10 glands from a separate tissue preparation of
one cat. C: phenylephrine (PHEP) responses in 4 species.
Mean peak responses to phenylephrine were normalized to the mean peak
responses to carbachol. No. of animals (n) per species is
shown above bars.
|
|
The small response to phenylephrine was unexpected based on prior
reports of strong secretion in the cat to
-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).
Secretion stimulated by isoproterenol.
The
-adrenergic agonist isoproterenol (10 µM) stimulated peak
gland secretion 1.8 ± 0.7 nl · min
1 · gland
1 (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).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 10.
Isoproterenol-stimulated gland secretion. Representative
secretion rates of isoproterenol (10 µM)-stimulated gland secretion
from a sheep are shown. Each closed circle denotes average basal and
stimulated secretion rates of 5 glands.
|
|
Inhibition of secretion with bumetanide.
In many epithelia, fluid secretion depends on secretion of
Cl
or HCO
. The
Na+-K+-2Cl
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).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 11.
HCO and Cl involvement
in gland secretions. A: inhibition (%) in the basal
secretion rate in the presence of 0.1 mM bumetanide (Bm) and/or
HCO replacement (HEPES). Data are means ± SE
from 25-39 glands and 4-7 separate tissue preparations from
3-4 sheep. *Significantly different from control,
P < 0.01. B: inhibition (%) in the
carbachol-stimulated secretion rate by bumetanide and HEPES. Data are
means ± SE from 24-33 glands and 4~5 separate tissue
preparations from 3-4 sheep. **Significantly different from
control, P < 0.01.
|
|
Inhibition of secretion by HCO
replacement.
HCO
-mediated fluid secretion also plays a role in
submucosal gland function (2), but the mechanisms for
HCO
transport are poorly understood, and no
specific, reliable inhibitors of the known HCO
transporters are available. Therefore, we assessed the contribution of
HCO
transport to gland mucus secretion by replacing
serosal HCO
with either 1 or 25 mM HEPES (see
METHODS). In response to HCO
removal
(Fig. 11), basal secretion was reduced to 45 ± 14% of control (39 glands from 3 sheep, P < 0.01), and peak
carbachol-stimulated secretion was reduced to 33 ± 10% of
control (33 glands from 3 sheep, P < 0.01).
Replacement of HCO
with either 25 mM HEPES or 1 mM
HEPES plus 24 mM NaCl produced similar inhibition of
carbachol-stimulated secretion, i.e., 60 ± 18% (10 glands in 2 sheep) and 75 ± 21% (16 glands in 2 sheep), respectively.
Inhibition of secretion by bumetanide plus
HCO
replacement.
Variable responsiveness to each of these inhibitors might be expected
if gland fluid secretion is mediated by a varying combination of
Cl
and HCO
transport. However, for
basal secretion, joint treatment with both bumetanide plus
HCO
replacement reduced secretion to 42 ± 15%
of control (35 glands in 2 sheep, P < 0.01, Fig. 11), which
did not differ significantly from the inhibition produced by
HCO
replacement alone (P > 0.8). In
contrast, for carbachol-stimulated secretion, the joint treatment
almost abolished secretion, leaving residual secretion of only 8 ± 2% of the control value (30 glands from 2 sheep, P < 0.01, Fig. 11). Although greatly reduced in amount, residual secretion
was still significantly greater than basal secretion (P < 0.05), suggesting the existence of at least one additional mechanism
for mucus secretion.
 |
DISCUSSION |
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
replacement. Basal and carbachol-stimulated secretion differed in several respects. Basal secretion was much smaller and more variable (in relative terms) than carbachol-stimulated secretion, it was less affected by inhibitors, and it diminished more
rapidly as a function of time postharvest. Because basal secretion was
lost in many glands that remained fully capable of secreting to
carbachol, both the decline and variability may be secondary to changes
in the numerous local mediators that affect gland secretion and are
presumably released during dissection and manipulation. Because gland
secretion in vivo is controlled by a host of powerful neural and
humoral factors, the physiological significance of the residual basal
secretion observed in these isolated preparations is uncertain.
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 · min
1 · gland
1 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
-mediated fluid secretion and that this reliance
varies across species.
Role of HCO
in gland secretion.
Replacement of HCO
with HEPES and the simultaneous
change of gassing from a 95% O2-5% CO2
mixture to air reduced basal secretion by 55% and cholinergically
stimulated peak secretion by 66%. The basis for this profound
inhibition of secretion is unknown. Basal short-circuit current in
Calu-3 cells, which is thought to be predominantly
HCO
dependent (8, 21), is also reduced
by >70% by replacement of HCO
with HEPES
(35). However, it is unlikely that secreted gland mucus
contains high levels of HCO
based on direct ion
measurements of uncontaminated mucus from cat submucosal glands, which
suggest that HCO
levels in mucus are similar to bath
values (29), and based on acidic pH measurements of airway
surface liquid in the ferret after carbachol stimulation (20). One possibility is that the fluid secreted by acinar
serous cells is indeed rich in HCO
, but most of the
HCO
is absorbed before the final mucus is secreted.
This and other possibilities need to be investigated.
In conclusion, we describe an optical method for quantifying
single-gland secretion rates with improved temporal resolution. The
method should be applicable to most gland-containing epithelia. We
introduced the method by focusing on secretion rates, but, because
secretions from each gland are isolated and protected from evaporation,
it is feasible to inject indicators for optical monitoring of pH, ionic
content, and viscosity. Gland ducts can subsequently be injected with
markers, followed by fixation and sectioning, to relate gland structure
and function. Single-gland methods are required if glands are
heterogeneous, as our initial results suggest. When used to compare
glands from normal and diseased tissues, these methods will allow the
testing of hypotheses about the contributions of gland dysfunction to
the pathogenesis of airway diseases.
 |
ACKNOWLEDGEMENTS |
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.
 |
FOOTNOTES |
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.
 |
REFERENCES |
1.
Ballard, ST,
Fountain JD,
Inglis SK,
Corboz MR,
and
Taylor AE.
Chloride secretion across distal airway epithelium: relationship to submucosal gland distribution.
Am J Physiol Lung Cell Mol Physiol
268:
L526-L531,
1995[Abstract/Free Full Text].
2.
Ballard, ST,
Trout L,
Bebok Z,
Sorscher EJ,
and
Crews A.
CFTR involvement in chloride, bicarbonate, and liquid secretion by airway submucosal glands.
Am J Physiol Lung Cell Mol Physiol
277:
L694-L699,
1999[Abstract/Free Full Text].
3.
Basbaum, CB,
Jany B,
and
Finkbeiner WE.
The serous cell.
Annu Rev Physiol
52:
97-113,
1990[ISI][Medline].
4.
Campbell, KH,
McWhir J,
Ritchie WA,
and
Wilmut I.
Sheep cloned by nuclear transfer from a cultured cell line.
Nature
380:
64-66,
1996[ISI][Medline].
5.
Choi, HK,
Finkbeiner WE,
and
Widdicombe JH.
A comparative study of mammalian tracheal mucous glands.
J Anat
197:
361-372,
2000[ISI][Medline].
6.
Corrales, RJ,
Nadel JA,
and
Widdicombe JH.
Source of the fluid component of secretions from tracheal submucosal glands in cats.
J Appl Physiol
56:
1076-1082,
1984[Abstract/Free Full Text].
7.
Davis, B,
and
Nadel JA.
New methods used to investigate the control of mucus secretion and ion transport in airways.
Environ Health Perspect
35:
121-130,
1980[ISI][Medline].
8.
Devor, DC,
Singh AK,
Lambert LC,
DeLuca A,
Frizzell RA,
and
Bridges RJ.
Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells.
J Gen Physiol
113:
743-760,
1999[Abstract/Free Full Text].
9.
Engelhardt, JF,
Yankaskas JR,
Ernst SA,
Yang Y,
Marino CR,
Boucher RC,
Cohn JA,
and
Wilson JM.
Submucosal glands are the predominant site of CFTR expression in the human bronchus.
Nat Genet
2:
240-248,
1992[ISI][Medline].
10.
German, VF,
Corrales R,
Ueki IF,
and
Nadel JA.
Reflex stimulation of tracheal mucus gland secretion by gastric irritation in cats.
J Appl Physiol
52:
1153-1155,
1982[Abstract/Free Full Text].
11.
German, VF,
Ueki IF,
and
Nadel JA.
Micropipette measurement of airway submucosal gland secretion: laryngeal reflex.
Am Rev Respir Dis
122:
413-416,
1980[ISI][Medline].
12.
Haws, C,
Finkbeiner WE,
Widdicombe JH,
and
Wine JJ.
CFTR in Calu-3 human airway cells: channel properties and role in cAMP-activated Cl
conductance.
Am J Physiol Lung Cell Mol Physiol
266:
L502-L512,
1994[Abstract/Free Full Text].
13.
Haxhiu, MA,
Cherniack NS,
and
Strohl KP.
Reflex responses of laryngeal and pharyngeal submucosal glands in dogs.
J Appl Physiol
71:
1669-1673,
1991[Abstract/Free Full Text].
14.
Hejal, R,
Strohl KP,
Erokwu B,
Cherniack NS,
and
Haxhiu MA.
Pathways and mechanisms involved in neural control of laryngeal submucosal gland secretion.
J Appl Physiol
75:
2347-2352,
1993[Abstract].
15.
Hejal, R,
Strohl KP,
Erokwu B,
Cherniack NS,
and
Haxhiu MA.
Effect of hypoxia on reflex responses of tracheal submucosal glands.
J Appl Physiol
78:
1651-1656,
1995[Abstract/Free Full Text].
16.
Inglis, SK,
Corboz MR,
and
Ballard ST.
Effect of anion secretion inhibitors on mucin content of airway submucosal gland ducts.
Am J Physiol Lung Cell Mol Physiol
274:
L762-L766,
1998[Abstract/Free Full Text].
17.
Inglis, SK,
Corboz MR,
Taylor AE,
and
Ballard ST.
Effect of anion transport inhibition on mucus secretion by airway submucosal glands.
Am J Physiol Lung Cell Mol Physiol
272:
L372-L377,
1997[Abstract/Free Full Text].
18.
Inglis, SK,
Corboz MR,
Taylor AE,
and
Ballard ST.
In situ visualization of bronchial submucosal glands and their secretory response to acetylcholine.
Am J Physiol Lung Cell Mol Physiol
272:
L203-L210,
1997[Abstract/Free Full Text].
19.
Jiang, C,
Finkbeiner WE,
Widdicombe JH,
and
Miller SS.
Fluid transport across cultures of human tracheal glands is altered in cystic fibrosis.
J Physiol (Lond)
501:
637-647,
1997[Abstract].
20.
Kyle, H,
Ward JP,
and
Widdicombe JG.
Control of pH of airway surface liquid of the ferret trachea in vitro.
J Appl Physiol
68:
135-140,
1990[Abstract/Free Full Text].
21.
Lee, MC,
Penland CM,
Widdicombe JH,
and
Wine JJ.
Evidence that Calu-3 human airway cells secrete bicarbonate.
Am J Physiol Lung Cell Mol Physiol
274:
L450-L453,
1998[Abstract/Free Full Text].
22.
Leikauf, GD,
Ueki IF,
and
Nadel JA.
Autonomic regulation of viscoelasticity of cat tracheal gland secretions.
J Appl Physiol
56:
426-430,
1984[Abstract/Free Full Text].
23.
Mariassy, AT,
and
Plopper CG.
Tracheobronchial epithelium of the sheep. I. Quantitative light-microscopic study of epithelial cell abundance, and distribution.
Anat Rec
205:
263-275,
1983[ISI][Medline].
24.
Meyrick, B,
and
Reid L.
Ultrastructure of cells in the human bronchial submucosal glands.
J Anat
107:
281-299,
1970[ISI][Medline].
25.
Meyrick, B,
Sturgess JM,
and
Reid L.
A reconstruction of the duct system and secretory tubules of the human bronchial submucosal gland.
Thorax
24:
729-736,
1969[ISI][Medline].
26.
Moon, S,
Singh M,
Krouse ME,
and
Wine JJ.
Calcium-stimulated Cl
secretion in Calu-3 human airway cells requires CFTR.
Am J Physiol Lung Cell Mol Physiol
273:
L1208-L1219,
1997[Abstract/Free Full Text].
27.
Nadel, JA,
and
Davis B.
Regulation of Na+ and Cl
transport and mucous gland secretion in airway epithelium.
Ciba Found Symp
54:
133-147,
1978[Medline].
28.
Nadel, JA,
and
Davis B.
Parasympathetic and sympathetic regulation of secretion from submucosal glands in airways.
Fed Proc
39:
3075-3079,
1980[ISI][Medline].
29.
Quinton, PM.
Composition and control of secretions from tracheal bronchial submucosal glands.
Nature
279:
551-552,
1979[ISI][Medline].
30.
Sasaki, H,
Sasaki T,
Shimura S,
and
Takishima T.
Effect of fenoterol on secretions of an isolated single submucosal gland from the trachea.
Respiration
50, Suppl2:
266-269,
1986[ISI][Medline].
31.
Sharma, P,
Dudus L,
Nielsen PA,
Clausen H,
Yankaskas JR,
Hollingsworth MA,
and
Engelhardt JF.
MUC5B and MUC7 are differentially expressed in mucous and serous cells of submucosal glands in human bronchial airways.
Am J Respir Cell Mol Biol
19:
30-37,
1998[Abstract/Free Full Text].
32.
Shen, BQ,
Finkbeiner WE,
Wine JJ,
Mrsny RJ,
and
Widdicombe JH.
Calu-3: a human airway epithelial cell line that shows cAMP-dependent Cl
secretion.
Am J Physiol Lung Cell Mol Physiol
266:
L493-L501,
1994[Abstract/Free Full Text].
33.
Shimura, S.
Signal transduction of mucous secretion by bronchial gland cells.
Cell Signal
12:
271-277,
2000[ISI][Medline].
34.
Shimura, S,
Sasaki T,
Sasaki H,
and
Takishima T.
Contractility of isolated single submucosal gland from trachea.
J Appl Physiol
60:
1237-1247,
1986[Abstract/Free Full Text].
35.
Singh, M,
Krouse M,
Moon S,
and
Wine JJ.
Most basal Isc in Calu-3 human airway cells is bicarbonate-dependent Cl
secretion.
Am J Physiol Lung Cell Mol Physiol
272:
L690-L698,
1997[Abstract/Free Full Text].
36.
Steel, DM,
Graham A,
Geddes DM,
and
Alton EW.
Characterization and comparison of ion transport across sheep and human airway epithelium.
Epithelial Cell Biol
3:
24-31,
1994[ISI][Medline].
37.
Trout, L,
Gatzy JT,
and
Ballard ST.
Acetylcholine-induced liquid secretion by bronchial epithelium: role of Cl
and HCO
transport.
Am J Physiol Lung Cell Mol Physiol
275:
L1095-L1099,
1998[Abstract/Free Full Text].
38.
Trout, L,
King M,
Feng W,
Inglis SK,
and
Ballard ST.
Inhibition of airway liquid secretion and its effect on the physical properties of airway mucus.
Am J Physiol Lung Cell Mol Physiol
274:
L258-L263,
1998[Abstract/Free Full Text].
39.
Ueki, I,
German VF,
and
Nadel JA.
Micropipette measurement of airway submucosal gland secretion. Autonomic effects.
Am Rev Respir Dis
121:
351-357,
1980[ISI][Medline].
40.
Widdicombe, JH,
Bastacky SJ,
Wu DX,
and
Lee CY.
Regulation of depth and composition of airway surface liquid.
Eur Respir J
10:
2892-2897,
1997[Abstract/Free Full Text].
41.
Wu, DX,
Lee CY,
Uyekubo SN,
Choi HK,
Bastacky SJ,
and
Widdicombe JH.
Regulation of the depth of surface liquid in bovine trachea.
Am J Physiol Lung Cell Mol Physiol
274:
L388-L395,
1998[Abstract/Free Full Text].
42.
Yamaya, M,
Finkbeiner WE,
and
Widdicombe JH.
Altered ion transport by tracheal glands in cystic fibrosis.
Am J Physiol Lung Cell Mol Physiol
261:
L491-L494,
1991[Abstract/Free Full Text].
Am J Physiol Lung Cell Mol Physiol 281(2):L458-L468
1040-0605/01 $5.00
Copyright © 2001 the American Physiological Society