RAPID COMMUNICATION
Airway surface fluid composition in the rat determined by capillary electrophoresis

Elizabeth A. Cowley, Karuthapillai Govindaraju, David K. Lloyd, and David H. Eidelman

Meakins-Christie Laboratories, Montreal Chest Institute Research Center, Royal Victoria and Montreal General Hospitals, McGill University, Montreal, Quebec, Canada H2X 2P2

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 HCO<SUP>−</SUP><SUB>3</SUB> 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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Table 1.   Composition of rat airway surface fluid as determined by capillary electrophoresis with conductivity detection

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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Fig. 1.   Dose-dependent increase in Na+ concentration ([Na+]; A) and Cl- concentration ([Cl-]; B) with increasing methacholine concentration ([MCh]; ng/g animal body wt). Values given are means ± SE (n = 3-11 animals). * Significance (P < 0.05) as determined by paired t-tests between the pre-MCh value and each subsequent dose.

Increases in the ASF K+ concentration after administration of MCh were not statistically significant at any dose.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 PO<SUP>2−</SUP><SUB>4</SUB> 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 HCO<SUP>−</SUP><SUB>3</SUB> within ASF, which is ~20% of that found in plasma. There has been only one other report of the amount of HCO<SUP>−</SUP><SUB>3</SUB> within ASF (2), and, in contrast, the authors reported HCO<SUP>−</SUP><SUB>3</SUB> values to be high in tracheal and bronchial canine ASF. This report did, however, indicate that the authors encountered technical difficulties measuring HCO<SUP>−</SUP><SUB>3</SUB> in such small quantities of fluid. Therefore, it may be that our value more accurately reflects the true value for HCO<SUP>−</SUP><SUB>3</SUB> within ASF or that significant species differences exist between the dog and the rat in terms of HCO<SUP>−</SUP><SUB>3</SUB> distribution. Unfortunately, we have been unable to measure the pH of rat ASF, which would give us additional information into the likely HCO<SUP>−</SUP><SUB>3</SUB> concentration.

We present here the first reported values for nitrite (NO<SUP>−</SUP><SUB>2</SUB>) and nitrate (NO<SUP>−</SUP><SUB>3</SUB>) within ASF. Significantly, both ions are at a higher concentration than are found in plasma (NO<SUP>−</SUP><SUB>2</SUB> at an approximately 3-fold higher concentration; NO<SUP>−</SUP><SUB>3</SUB> 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 NO<SUP>−</SUP><SUB>2</SUB> and NO<SUP>−</SUP><SUB>3</SUB>, 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.

    ACKNOWLEDGEMENTS

We thank Dr. Paul Linsdell for useful discussion of this manuscript.

    FOOTNOTES

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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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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