Meakins-Christie Laboratories, Montreal Chest Institute Research Center, Royal Victoria and Montreal General Hospitals, McGill University, Montreal, Quebec, Canada H2X 2P2
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ABSTRACT |
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The apical surface
of respiratory epithelial cells is covered by a thin layer of
low-viscosity fluid termed airway surface fluid (ASF), about which
relatively little is known. We collected samples of ASF from
anesthetized rats, which were then analyzed using capillary
electrophoresis, a method that enables extremely small quantities of
fluid to be analyzed. We found values for Na+ (40.57 ± 3.08 mM),
K+ (1.74 ± 0.36 mM), and
Cl (45.16 ± 1.81 mM),
indicating that this fluid is hypotonic compared with rat plasma. In
contrast, the concentrations of nitrite and nitrate within ASF were
higher than reported plasma values. Additionally, intravenous
administration of the cholinergic agonist methacholine (MCh) resulted
in a dose-dependent increase in the concentration of
Na+ and
Cl
within the ASF. This
increase is ~50% in these ions after a dose of 100 ng MCh/g body wt.
This animal model, together with this microanalytical technique, may be
useful for investigating the in vivo regulation of ASF composition.
airway epithelium; microanalysis; methacholine
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INTRODUCTION |
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THE CONDUCTING AIRWAYS of the respiratory tract are covered by a thin layer of fluid termed airway surface fluid (ASF). Traditionally, this fluid was thought to provide a low-viscosity medium in which cilia can beat, thus enabling the mucociliary clearance apparatus to transport particulate matter toward the oropharynx. Therefore, although the quantity of ASF may affect ciliary beating efficiency (13) and additionally may play a role in the hydration of mucus and its rheology (21), this was thought to be the extent of its physiological role. Furthermore, in terms of its composition, ASF was believed to be simply an isotonic transudate of plasma. It is becoming increasingly apparent, however, that not only the quantity, but also the composition, of this fluid may play a fundamental role in pulmonary defense mechanisms (17).
A report by Smith et al. (20) proposed that ASF contains a potent
bactericidal factor important in maintaining a sterile environment
within the healthy lung. Furthermore, Smith et al. (20) hypothesized
that a high ionic strength within the ASF would lead to inhibition of
this bactericidal factor. This hypothesis is the first to suggest a
mechanism by which the defective epithelial Cl conductance seen in
cystic fibrosis could account for the extensive bacterial colonization,
and subsequent lung pathology, seen in this disease. Unfortunately,
however, previous reports describing the composition of ASF in normal
airways have been somewhat contradictory, with the result that little
is known about the normal ionic composition and regulation of this
fluid. This paucity of information reflects the tremendous difficulties
associated with collection and subsequent analysis of such small,
relatively inaccessible fluid samples.
The composition of ASF had been determined in earlier studies by
indirect methods, namely the collection of fluid onto filter paper and
its subsequent extraction for analysis by flame photometry (2) or
energy-dispersive X-ray spectroscopy (9, 10). In this report, we
describe the collection of extremely small (~100 nl) samples of ASF
from rats directly into capillary tubing. Analysis was then performed
using capillary electrophoresis (CE) with conductivity detection, a
method that we have recently applied to the analysis of rat ASF (6).
Using this technique, we are able to analyze extremely small fluid
samples, making it ideal for the analysis of ASF. Here, we report
values for the major anions and cations in rat ASF. Additionally, we
report that intravenous administration of the cholinergic agonist
methacholine (MCh) results in increases in
Na+ and
Cl concentrations within
the ASF. We believe, therefore, that this system may prove useful for
future studies into ASF regulation and for the role of ASF in host
defense mechanisms.
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MATERIALS AND METHODS |
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Male Fisher rats (350-400 g; Harlan Sprague Dawley, Indianapolis, IN) were sedated with xylazine (0.08 ml/100 g body wt) and then were anesthetized with pentobarbital sodium (0.053 ml/100 g body wt) injected intraperitoneally. An intubation tube (1.67 mm ID, 2.42 mm OD, length 6 cm) was then inserted, and the animal was placed in a supine position. A 10-cm length of polyethylene tubing (PE-10 tubing, 0.28 mm ID, 0.61 mm OD, length 10 cm; Becton-Dickinson, Sparks, MD) was then introduced into the intubation tube so that 1 cm of this sampling capillary projected from the top of the intubation tube. Fluid samples were therefore collected from ~0.5 cm below the level of the carina and from the same level in each animal. The sampling capillary remained in the animal for a period of 5 min, after which it was carefully removed, and the ASF was analyzed immediately. The volume of ASF typically collected in a 5-min period was ~100 nl.
ASF analysis was performed using two methods of CE. The first, CE with conductivity detection, allows a variety of ions to be detected. In this case, analysis was performed using a Crystal CE system with a Concap capillary and Contip conductivity sensor from ATI Unicam (Boston, MA), modified to allow direct injection of ASF samples (~5 nl sample/injection; 3-5 samples/animal). Samples were thus loaded into the CE machine by automatic injection, avoiding the need for manual manipulation of such small volumes. Data were collected with an integrator (model SP4600; Spectra-Physics, San Jose, CA), and data storage and manipulation were performed with Spectra-Physics Winner software (see Ref. 6 for details of capillary electrophoretic setup).
Anion analysis was performed in a buffer containing 100 mM
2-(N-cyclohexylamino)ethanesulfonic
acid and 40 mM lithium hydroxide (Sigma Chemical, St. Louis, MO), and
2-propanol (Fisher Scientific, Nepean, ON, Canada) at 92:8
(vol/vol), pH 9.3, with 80 µM spermine (Sigma Chemical) as an
electroosmotic flow modifier. Cation analysis was performed in a buffer
containing 100 mM
2-(N-morpholino)ethanesulfonic acid,
100 mM DL-histidine, and 20 mM -hydroxyisobutyric acid, pH 5.6 (Sigma Chemical). All buffers were prepared fresh daily in
deionized water (Milli-Q unit; Millipore, Montreal, PQ, Canada), degassed by sonication, and filtered through 0.45-µm membrane filters
(Gelman Sciences, Montreal, PQ, Canada). For anion analysis, the
separation potential was
2,78 V/cm [current (I) = 9.5 µA], whereas for cations it was 222 V/cm (I = 12.5 µA). Analysis was carried out at 25°C.
For the series of experiments involving intravenous administration of
the cholinergic agonist MCh (ICN Chemicals, Mississauga, ON, Canada), a
catheter was inserted into the jugular vein, and ASF was collected via
an intubation tube as described above. MCh was then administered
intravenously at concentrations between 10 and 100 ng MCh/g body wt, as
this agent has previously been reported to either decrease ASF
Cl concentrations (2) or to
have no effect (10).
A second method of CE was used to analyze the samples of ASF collected
after MCh administration. Here, CE with indirect ultraviolet (UV)
detection was chosen as this technique was already well established in
our laboratory (22), therefore allowing us to analyze several samples
per day. Briefly, analysis was carried out using a CE unit (270 A) from
Applied Biosystems (Foster City, CA), and three to five samples were
analyzed again per animal. The analysis of Cl was performed using a
buffer containing 5 mM sodium chromate with 1.50 mM hexamethonium
hydroxide, adjusted to pH 7.0 with 1 M
H2SO4.
A vacuum of 17 kPa was applied for 0.5 s for sample injection (~1
nl), and the separation potential was
347 V/cm (I = 13 µA)
with a detection wavelength of 273 nm. For the cation (Na+ and
K+) analysis, a buffer
containing 10 mM imidazole with 8% (vol/vol) 2-propanol, adjusted to
pH 3.5 with 1 M HCl, was used. The separation potential was 347 V/cm (I = 11 µA) with detection at 214 nm. The capillary oven temperature was
set at 30°C for both anion and cation analysis.
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RESULTS |
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ASF composition. Calibration was carried out by taking the peak area under the curve of known concentrations of the ion in question. This method was linear over the concentration range of interest, and regression coefficients between 0.994 and 0.999 were obtained in all cases.
Values for the ionic composition of rat ASF determined by CE with
conductivity detection are given in Table
1. The values obtained for
Na+,
K+,
Ca2+,
Mg2+,
Cl, and
are all lower than reported
values for either rodent or human plasma, demonstrating that overall rat ASF is hypotonic compared with plasma. Values for
Na+ and
Cl
within plasma were also
measured using CE and were found to be 136 mM for
Na+ and 100 mM for
Cl
.
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To address the question of whether the presence of proteins within ASF could potentially effect our results, we compared repeated measurements of Na+ and K+ standards made up in water with standards made up in a proteinaceous medium (albumin at 30.5 mg/ml). Over 10 measurements made in water, the error was found to be 2.5% for Na+ and 6% for K+. Five measurements were made in albumin, and the errors were determined to be 9% for Na+ and 8% for K+.
MCh studies. The effects of increasing
intravenous administration of MCh on
Na+ and
Cl concentrations are shown
in Fig. 1. There is a dose-dependent increase in the concentration of both of these ions with increasing MCh. The Na+ concentration within
the ASF increased by 49.9% (45.98 ± 3.34 mM before the addition of
MCh to 68.92 ± 7.56 mM after 100 ng MCh/g body wt). This increase
in Na+ was statistically
significant (P < 0.05)
using paired t-tests between baseline
and 25, 50, and 100 ng MCh/g body wt. Similarly, Cl
increased 49.8% (from
40.02 ± 1.2 mM to 59.97 ± 1.10 mM after 100 ng MCh/g body wt).
This increase was statistically significant (P < 0.05) between baseline and 50 and 100 ng MCh/g body wt.
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Increases in the ASF K+ concentration after administration of MCh were not statistically significant at any dose.
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DISCUSSION |
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The results presented here demonstrate that the technique of CE can be used for detailed analysis of small quantities of ASF. Use of CE with conductivity detection allows for the resolution of a greater number of ionic species at a lower limit of detection than CE with indirect UV light (22), and, importantly, this analytical technique does not depend on the collection onto, and subsequent extraction from, filter paper. Our analysis found rat ASF to be considerably hypotonic compared with plasma, a finding in agreement with several previous reports of ASF composition from different species (5, 9, 10). These data confirm the notion that the ionic composition of ASF is actively regulated.
Our finding that rat tracheal ASF is hypotonic does, however,
contradict previous reports that dog (2) and ferret (18) tracheal ASF
are hypertonic. There are several reasons that may account for this
apparent discrepancy. First, it is possible that differences in ASF
composition may occur between different species. Earlier reports of ASF
composition have used larger mammals such as dogs (2), ferrets (18), or
horses (10) as models, all of which possess large numbers of submucosal
glands throughout the proximal airways. Rats have far fewer of these
glands, concentrated in the trachea. Therefore, it is possible that, if
submucosal gland secretions contribute significantly toward the
production of ASF, electrolyte composition in the rat may differ from
those species that have abundant submucosal glands. Second, reports of
bronchial ASF composition indicate that it has lower levels of
Na+ and
Cl compared with plasma (2,
9), which may also suggest the possibility that regional differences in
salt concentration exist across the airway tree. Finally, the major
advantage of our technique is that ASF samples are collected in vivo
and that unmanipulated samples are then analyzed directly. Therefore,
although the actual molar values that we measure are considerably lower
than those previously reported (5, 9), this may well be accounted for by differences in the analytical techniques used or by differences in
the method of fluid collection.
The mechanism by which a hypotonic ASF is produced remains to be
elucidated. One possibility is that ASF arises from an initially isotonic secretion. Active reabsorption of
Na+ and
Cl across the apical
membrane of airway epithelial cells could then occur, presumably
through the amiloride-sensitive epithelial
Na+ channel and the CFTR
Cl
channel, thus resulting
in low extracellular Na+ and
Cl
. This
scenario is reminiscent of the situation that occurs in the sweat duct,
whereby secretion of an isotonic fluid in the proximal duct is followed
by reabsorption of electrolytes in excess of water in the distal
tubule, thus producing a hypotonic fluid (16). However, this hypothesis
requires the respiratory epithelium to be relatively impermeable to
water. Given the recent observations that the respiratory system
contains specific water channels (14), such impermeability may not to
be the case, and other as yet unidentified forces may act to retain
water on the apical surface of epithelial cells.
We report that intravenous administration of the cholinergic agonist
(MCh) induced an increase in the
Na+ and
Cl concentration within
ASF. Joris and Quinton (10) reported that administration of MCh had no
effect on the composition of the fluid that they collected from in
vitro samples of horse trachea. In contrast, Boucher et al. (2)
reported a decrease in tracheal Na+ concentration along with an
increase in Cl
using in
vivo dog bronchi. Among the many reported effects of cholinergic
stimulation are increases in active ion transport (19) and the
production of fluid (12) from submucosal glands. It is possible
therefore that the increases we see in
Na+ and
Cl
concentration with
increasing MCh may reflect either an increased transport of these ions
or an overall increase in the amount of fluid secreted from submucosal
glands. Indeed, after MCh administration, we did note that fluid was
easier to collect and that volumes tended to be greater, consistent
with increased glandular secretion. Although this study did not address
the potential sources of basal ASF in the rat, it is likely that the
fluid we collect after cholinergic stimulation originates at least in
part in the submucosal glands.
We found values for K+,
Ca2+, and
in ASF lower than those
reported in earlier studies and also lower than the values described
for plasma. In particular, all studies have reported a high
K+ concentration within ASF across
a variety of species. One possibility is that previous reports of ASF
composition that involved the transfer of liquid to filter paper (2, 9,
10) induced some damage to surface cells and the consequent release of
intracellular K+. Our introduction
of an extremely small-diameter capillary tube may be less damaging to
the epithelial cells, thus permitting us to record a more physiological
range of values for K+. Our
finding of low K+ suggests the
possibility that there may be some active absorptive mechanism for this
ion across the rat tracheal epithelium. The respiratory epithelium has
not been described as capable of
K+ reabsorption; indeed, it has
been suggested that some airway epithelia may actually secrete
K+ (3, 4). Although 4th-6th
generation bronchi of sheep (4) and dog (3) secreted small amounts of
K+, the trachea did not. To date,
there have been no reported measurements of
K+ flux across the rat tracheal
epithelium, and the possibility of an active uptake mechanism cannot be
excluded.
We report a value of 5.92 ± 0.93 mM for
within ASF, which is ~20% of
that found in plasma. There has been only one other report of the
amount of
within ASF (2), and, in
contrast, the authors reported
values to be high in tracheal and bronchial canine ASF. This report did, however, indicate that the authors encountered technical difficulties measuring
in such
small quantities of fluid. Therefore, it may be that our value more
accurately reflects the true value for
within ASF or that significant
species differences exist between the dog and the rat in terms of
distribution. Unfortunately, we
have been unable to measure the pH of rat ASF, which would give us
additional information into the likely
concentration.
We present here the first reported values for nitrite
() and nitrate
(
) within ASF. Significantly, both
ions are at a higher concentration than are found in plasma
(
at an approximately 3-fold higher
concentration;
at 500 times the
concentration seen in plasma). Nitric oxide is present within the
exhaled air of both humans and rodents (7) and is synthesized within
the lungs via the action of nitric oxide synthase by a number of cells, including epithelial cells (1) and macrophages (8). After its
synthesis, nitric oxide is rapidly broken down to a variety of
secondary products, including
and
, and it seems probable that the
levels of these species we record in the ASF reflect local production
of nitric oxide by cells containing a constitutively expressed nitric
oxide synthase. Our technique may potentially demonstrate a new
approach enabling nitric oxide production within the lung to be
investigated.
The values that we report were recorded from rats anesthetized with pentobarbital sodium, an agent suggested to exert an anticholinergic effect. However, studies that we also carried out using the anesthetic agent ethyl carbamate (urethan; data not shown) revealed no difference in either the quantity or composition of ASF collected compared with study results using pentobarbital sodium. It is likely, therefore, that pentobarbital sodium has little or no effect on ASF composition.
In conclusion, we have demonstrated that it is possible to collect small amounts of ASF from rats in vivo and that analysis of this fluid by CE gives highly reproducible values for a wide variety of ions. Our report that rat ASF is hypotonic compared with plasma is consistent with an earlier report of ASF from normal human subjects (9). This would be compatible with the hypothesis proposed by Smith et al. (20), as the production of a hypotonic solution to line the airways could provide a medium enabling the proposed antibacterial factor to function optimally. Although much remains to be elucidated about the production and regulation of this fluid, the development of animal models is an important step toward this goal. Furthermore, our report that the composition of ASF can be manipulated pharmacologically presents us with a potential system with which to investigate the role of ASF in host defense.
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ACKNOWLEDGEMENTS |
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We thank Dr. Paul Linsdell for useful discussion of this manuscript.
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FOOTNOTES |
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This work was supported by the Canadian Cystic Fibrosis Foundation, the Respiratory Health Network of Centres of Excellence, the Montreal Chest Institute Research Centre, and the J. T. Costello Foundation.
D. H. Eidelman and D. K. Lloyd were the recipients of Chercheur-Boursier awards of the Fonds de la Recherche en Santé du Québec.
Present address of D. K. Lloyd: Du Pont Merck Pharmaceutical Company, Analytical R & D, Experimental Station E353, PO Box 80353, Wilmington, DE 19880-0353.
Address for reprint requests: D. H. Eidelman, Meakins-Christie Laboratories, Montreal Chest Institute Research Centre, Royal Victoria and Montreal General Hospitals, McGill University, 3626 St. Urbain St., Montreal, PQ, Canada H2X 2P2.
Received 25 February 1997; accepted in final form 4 August 1997.
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