Introduction

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Cystic fibrosis (CF) is an inherited disease associated with the accumulation of thick and purulent airway secretions (1). Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein are responsible for abnormal ion transport leading to complex pathophysiology of mucociliary function with impaired mucus clearance in CF lung disease (2). Recent attention has been focused on the fact that chloride secretion in CF epithelial cells can be stimulated through Ca²⁺-activated, alternative pathways that do not require the CFTR protein (3). In preliminary studies, we found that mice, particularly the C57BL/6 strain, have a substantial tracheal mucus gland located immediately distal to the larynx (6). Further, preliminary data suggest that the secretion of tracheal mucus in CFTR-knockout mice (7) is unresponsive to secretagogue activation by methacholine (MCh), whose action depends at least in part on functional CFTR protein (8). This finding is consistent with the fact that in mouse airways, chloride secretion through non-CFTR pathways is known to compensate for the lack of CFTR-dependent secretion (9, 10).

We hypothesized that the mucus in CFTR-knockout mice is quasinormal because of the alternative, non-CFTR pathways to chloride secretion, and when these pathways are blocked, this could lead to the development of mucociliary dysfunction. Previous studies have focused on the effectiveness of chloride secretagogues, particularly on triphosphate nucleotides such as adenosine triphosphate (ATP) and uridine 5'-triphosphate (UTP) (11, 12). UTP promotes transepithelial chloride transfer, which we confirmed both by its effect on potential difference (PD), as well as on the chloride ion content of the collected secretion (12). Therefore, the purpose of this project was to compare the mechanism of action of MCh and UTP on tracheal mucus secretion in mice, which could relate to the presence of constitutive non-CFTR-dependent chloride channels. Also the effect of agonists that interact with P₂ receptors in mice airways was evaluated by using the chloride channel inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS). DIDS has been shown to inhibit the chloride secretion induced by ATP (13). Both DIDS and the CFTR blocker 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) (14) were used as pretreatments for MCh.

	Materials and Methods

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Animals

Healthy C57BL/6 mice weighing 18 to 20 g were anesthetized with an intraperitoneal injection of 8 mg/kg xylazine and 70 mg/kg pentobarbital. During the mucus collection procedure, the animals were maintained in a chamber humidified by Ringer aerosol.

Mucus Collection

Mucus was obtained from the anesthetized mice by performing a tracheotomy and inserting a small polyethylene catheter (PE10; outer diameter, 0.61 mm; inner diameter, 0.28 mm) retrograde toward the larynx. The catheter was left in place for 1.5 h, and then withdrawn and placed immediately in a test tube containing paraffin oil to minimize evaporation loss. Adherent mucus was scraped off the catheter and transferred to an analytical container inserted into a magnetic microrheometer. When necessary, tracheal samples from two matched mice were combined to achieve samples sufficiently large to analyze.

After control mucus collection, a new catheter was inserted just before administration of the agent being tested. Collection times were 15 min for MCh, 30 min for DIDS+ MCh and NPPB+MCh, and 45 min for UTP and DIDS+ UTP. These collection times were chosen to provide equivalent opportunity to collect sufficient volume for rheologic analysis of each treatment.

Transepithelial Potential Difference

PD measurements relate to the ion content of mucus (15); they also help to assess the integrity of the epithelium because alterations of cellular and paracellular pathways contribute to PD. PD was measured by using two flexible microelectrodes (agar bridges of PE10 size) saturated with KCl connected to calomel half cells, one for reference and the other for testing. These were connected to the high impedance input of a grounded pH meter (Accumet 925; Fisher Scientific, Pittsburg, PA). The reference probe was inserted subcutaneously into the hind leg of the anesthetized mouse in a supine position. The measurement probe was introduced carefully into the upper trachea and placed in contact with the epithelium within 1 to 2 mm of the larynx. Care was taken to position the measurement tip to make the slightest contact with the mucosal surface. The PD value was recorded when a steady state value was achieved and remained stable for at least 30 s.

Rheologic Measurements

This technique consists of analyzing the bulk viscosity and elasticity of microliter quantities of mucus (16). A 50- to 100-µm steel ball was positioned in an approximately 1-µl sample of mucus, and the motion of this sphere under the influence of an oscillating electromagnetic field gradient was used to determine the rheologic properties of the mucus. The image of the steel ball was projected via a microscope onto a pair of photocells, whose output voltage was amplified and transmitted to an oscilloscope. By plotting the displacement of the ball against the magnetic driving force, the viscoelastic properties of the mucus were ascertained (17).

The main parameter of mucus viscoelasticity determined was the rigidity index or mechanical impedance, i.e., G*, reported here on a log scale, expressing the vector sum of "viscosity + elasticity," over the frequency range 1 to 100 rad/s. The rheologic properties can be used to predict the effectiveness of mucus in clearance, both by ciliary action and for clearance by airflow interaction (18).

Drug Administration

MCh (acetyl--methylcholine chloride, A2251; Sigma Chemical, St. Louis, MO) and UTP (trisodium salt, type VI, Sigma U6875) solution was freshly prepared at a concentration of 10² M in nonlactated Ringer's solution. The chloride channel blocker DIDS (disodium salt, 80-90%, Sigma D3514) and the CFTR antagonist NPPB (N-150; Research Biochemicals Inc., Natick, MA) were also freshly prepared, at a concentration of 10² M. After a 1.5-h control period used to collect sufficient mucus without stimulation, either MCh solution, UTP, DIDS, or NPPB followed 10 min later by UTP or MCh was instilled in 5 µl volume external to the larynx. Care was taken to ensure that the instillate did not enter the tracheal stoma.

In this study, 18 mice received MCh, 16 mice received UTP, 10 mice received DIDS+UTP, 9 mice received DIDS+MCh, and 10 mice received NPPB+MCh. Mucus was collected and PD was measured before and after each drug treatment, except for DIDS or NPPB alone, where only PD was measured during the short interval before administration of UTP or MCh.

Statistical Analysis

The significance of the results was assessed by analysis of variance for repeated measures using the statistical program StatView (Abacus Concepts, Berkeley, CA) for Macintosh. All results are presented as mean ± standard error of the mean (SEM) unless otherwise stated. Between treatment comparisons for PD, logG*, and mucus collection rate were made using paired t tests. The level of significance was set at 5%.

	Results

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Mucus samples sufficient for rheologic analysis were collected from 53 of 63 mice in control conditions and from 55 of 63 mice after treatment. PD measurements were made successfully in all but three mice.

As indicated in Figure 1, there was a significant shift to higher negative values for PD after MCh stimulation (21.3 ± 2.0 mV for MCh versus 14.1 ± 1.6 mV for control, P = 0.0004) and UTP stimulation (25.4 ± 2.5 mV for UTP versus 19.2 ± 1.9 mV for control, P = 0.028). Instillation of DIDS lowered PD (less negative) (from 15.2 ± 2.9 to 12.0 ± 1.9 mV, P = 0.056, nonsignificant). Application of UTP did not alter the tracheal PD further, but compared with baseline, the combination of DIDS+UTP showed a significant decrease (from 15.2 ± 2.9 to 12.0 ± 2.2 mV, P = 0.014). Application of MCh after either DIDS or NPPB did not change PD significantly. Overall, in 19 mice, administration of DIDS alone resulted in a significant decrease in tracheal PD compared with untreated control (13.5 ± 1.2 versus 15.9 ± 1.6 mV, P = 0.027).

View larger version (34K): [in this window] [in a new window]   Figure 1.   (A) The effects on the transepithelial potential difference in mouse trachea of paired treatments of 102 M of MCh (n = 18), MCh preceded by DIDS (n = 9), or MCh preceded by NPPB (n = 10), compared with their respective controls. Values are plotted as the mean ± SEM. (B) PD in mouse trachea of paired treatments of 102 M of UTP (n = 16) or UTP preceded by DIDS (n = 10), compared with their respective controls. The PD was significantly more negative for MCh and for UTP, and less negative for DIDS+UTP. Overall, in 19 mice treated with DIDS alone, PD decreased from 15.9 ± 1.6 to 13.5 ± 1.2 mV (P = 0.027).

At the same time, there was a significant decrease in logG* at 10 rad/s with MCh stimulation compared with control (from 2.99 ± 0.14 to 2.54 ± 0.09, P = 0.003). When MCh administration was preceded by DIDS, there was a diminished but still significant decrease in logG* (from 2.53 ± 0.13 to 2.34 ± 0.13, P = 0.034). Administration of the combination NPPB+MCh left logG* unchanged from its control (2.35 ± 0.13 for NPPB+MCh versus 2.42 ± 0.12 for control). Fourteen of 16 C57BL/6 mice responded to UTP in terms of PD becoming more negative. Of these, there was a significant difference in logG* before and after UTP (2.29 ± 0.10 for UTP versus 2.57 ± 0.10 for control, P = 0.050), whereas there was no change in logG* when the UTP administration was preceded by DIDS (2.57 ± 0.19 for DIDS+UTP versus 2.73 ± 0.16 for control) (Figure 2). The changes in mucus rheology seen at 1 and 100 rad/s measurement frequency were similar to those reported for 10 rad/s. There were no significant differences in the loss tangent ("viscosity/elasticity") associated with mediator administration.

View larger version (27K): [in this window] [in a new window]   Figure 2.   Mucus viscoelasticity, measured in terms of logG* at 10 rad/s, for the five paired treatments referred to in Figure 1. MCh produced the most significant decrease in logG* whereas UTP had a smaller yet significant effect on mucus viscoelasticity. DIDS treatment reduced the magnitude of decrease in logG* owing to MCh. By contrast, DIDS mitigated the effect of UTP as revealed by the logG* value, which is not significantly different from control. NPPB blocked the change in mucus viscoelasticity owing to MCh. Values are presented as mean ± SEM.

The mean weight of control mucus samples was 0.23 mg; after MCh administration, mean sample size was 0.70 mg, and after UTP it was 0.26 mg. The weight of mucus collected per hour per mouse was 0.19 ± 0.09 mg/h, whereas after MCh, the mucus collection rate increased to 2.83 ± 0.78 mg/h (P = 0.009). After DIDS+MCh, the mucus collection rate increased from 0.12 ± 0.01 to 0.61 ± 0.17 mg/h (P = 0.018), and after NPPB+MCh, it increased from 0.23 ± 0.06 to 0.91 ± 0.26 mg/h (P = 0.047). There was no significant difference in collection rate before and after UTP (0.17 ± 0.03 versus 0.30 ± 0.08 mg/h), although there was a tendency to increase (P = 0.056). In contrast, there was a lack of change observed for the group treated with DIDS+UTP (0.16 ± 0.04 versus 0.19 ± 0.04 mg/h) (Figure 3). All the changes induced by UTP treatment appeared to be blocked by DIDS.

View larger version (17K): [in this window] [in a new window]   Figure 3.   A graph of mucus collection rate (MCR) for the five paired treatments referred to in Figure 1. MCh induced a significant increase (P = 0.009) in MCR, and UTP showed a near-significant increase (P = 0.056), compared with the corresponding controls. DIDS+MCh treatment led to a smaller yet still significant increase in MCR, compared with MCh alone, whereas DIDS+UTP treatment did not show an effect on MCR. The increase in MCR after NPPB+MCh treatment was significant but smaller than without NPPB.

There were significant variations between the mean control values of PD and logG* among the different groups of mice studied. This was also the case within the control data set where a significant correlation between baseline logG* and tracheal PD was observed. This is illustrated in Figure 4. A significant correlation between mucus collection rate and logG* was also noted, as illustrated in Figure 5.

View larger version (15K): [in this window] [in a new window]   Figure 4.   A plot of logG* versus PD for the control data from four series of mice as indicated. The positive relationship (P = 0.026) accounts for some of the variation in mean control values of logG* and PD among the experimental groups.

View larger version (14K): [in this window] [in a new window]   Figure 5.   The relationship between tracheal mucus collection rate (in mg/h) and mucus viscoelasticity (represented by logG* at 10 rad/s) for pretreatment controls. The negative slope is significant (P = 0.027), indicating that the higher collection volumes are associated with less rigid mucus.

	Discussion

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One of the limitations of murine models of airway disease is the difficulty in collecting sufficient mucus or airway surface fluid for conventional analysis. We have found that it is possible to harvest tracheal mucus from laboratory mice in quantities sufficient for rheologic analysis by means of judicious choice of sampling site, prolonged collection times, and further miniaturization of the magnetic rheometer technique for rheologic testing (19). The choice of sampling site (just below the larynx) and the use of C57 mice were based on a suggestion from Dr. Barbara Grubb (University of North Carolina, Chapel Hill, NC) and confirmed by observations by Cowley and coworkers (6) that the mucus glands in C57BL/6 mice are relatively large compared with other inbred strains. Using this new technology, we reported that tracheal mucus in CFTR-knockout mice is not abnormal, but the animals are unresponsive to the secretagogue action of methacholine (7). As seen in the present study, mouse tracheal mucus, although small in quantity, is nevertheless rheologically similar to dog (16), ferret (20), and normal human mucus (21).

In this investigation, further miniaturization of the magnetic microrheometer technique was required in order to deal with the very small samples of mucus (~ 0.3 mg) that could be obtained from mouse trachea. Previously the magnetic rheometer method needed a minimum of 1 mg, using steel microspheres of ~ 100 µm (16). In the present investigation, we found that 0.3-mg samples could be analyzed by using smaller steel balls (for example, 70 µm), keeping the ratio of ball size to linear sample dimensions relatively constant, at about 1:10 or less.

Takahashi and colleagues (22) have studied the tracheal epithelial potential difference in several strains of mice, including C57BL/6, for which they reported a range of 12 to 15 mV (lumen negative), with initial vales of 15 to 17 mV. Although we saw a wider range of baseline PD than reported by these investigators, it is noted that our observed range overlaps that reported by Takahashi and coworkers (22) in about 80% of the animals studied (Figure 4). Our experience in dogs (23) has indicated that a more negative and stable PD is obtained when the agar-filled electrode is allowed to develop a filament of mucus as the contact with the epithelial layer. Perhaps the paucity of the mucous layer in mouse trachea adds to the variability in this measurement. We did observe that the variations in baseline PD correlate with the baseline variations in mucus viscoelasticity (see Figure 4), suggesting that fluctuations in transepithelial ion transport implied by the variations in PD contribute to the rheologic properties of the mucus gel by influencing mucus hydration. The negative correlation between mucus collection rate and logG* within controls (Figure 5) lends credence to the concept that this is tracheal mucus that we are sampling and not a serous exudate, and that the larger volumes of mucus are associated with increased transepithelial water movement.

Tracheal PD was altered significantly to higher negative values after either MCh or UTP stimulation (mean increase of 7.2 mV for MCh and 6.2 mV for UTP). At the same time, there was a significant decrease in logG* at 10 rad/s with MCh administration compared with control (from 2.99 ± 0.14 to 2.54 ± 0.09, P = 0.003), and a more modest decrease in logG* after UTP administration (2.29 ± 0.10 for UTP versus 2.57 ± 0.10 for control, P = 0.05) in the 14 of 16 mice that responded in terms of PD. In contrast to the qualitatively similar effects on PD and tracheal mucus rheology, the effect of UTP on the volume of mucus secreted was trivial, compared with the order-of-magnitude increase in mucus collection rate seen with MCh administration.

It has previously been established that PD increases, i.e., becomes more negative, with the use of UTP (4, 12). However, no comparison of mucus effects between MCh and UTP has been described before. Jiang and colleagues (24) demonstrated that the CFTR-dependent Cl channel cannot be stimulated by UTP, but the Ca²⁺-dependent Cl channel can be stimulated by UTP through the purinergic P₂ receptors. The chloride channels involved are probably Ca²⁺-dependent channels, especially because other chloride channels that would be affected by DIDS are not voltage dependent (3). We have reported that UTP administration in frog palate stimulated the output of both mucin and water, and overproduction of epithelial fluid led to a reduced rate of mucociliary transport (12), in contrast to the improvement seen with amiloride (25). In this study, there was a strong correlation between the changes in PD and the changes in mucus viscoelasticity, supportive of a commonality in the relationship between transepithelial chloride transport and hydration of the mucous gel, despite the differences in specific mechanisms.

In the current study, an increase in PD and a decrease in mucus rigidity were demonstrated with UTP. The fact that PD changes and mucus alterations were prevented after DIDS pretreatment suggests that these changes are associated with the chloride channel owing to P₂ receptors in mice, because cyclic adenosine monophosphate (cAMP)- mediated, CFTR Cl channels are resistant to inhibition by stilbene derivatives (26). It is likely that the population of Ca²⁺-dependent Cl channels stimulated by UTP P₂ receptors in mice is limited, compared with that of the mixed population of chloride channels responding to secretagogues such as MCh. Although in mouse trachea the hypersecretion of mucus due to UTP is trivial in comparison with MCh, a similar rheologic change is seen with both mediators, suggesting that their relative effects on water movement are stronger than their effects on mucin output. Shimura and colleagues (27) have shown that P₂ receptor stimulation results in both electrolyte and mucin secretion. Furthermore, ATP-induced secretion in isolated feline tracheal submucosal glands is enhanced by isoproterenol, a -receptor stimulator, via intracellular interaction with cAMP. It may be that inherent variations between animal species in -adrenergic function or intracellular cAMP levels affect the degree to which UTP induces the secretion of mucous glycoproteins.

The action of MCh is complex. Yamaya and associates (28), in a study of secretion in cultured tracheal gland cells, report that in non-CF gland cells, the ion transport response to MCh is mainly transient, reflecting an increase in Cl secretion. Assuming these cells, like colonic epithelial cells, have mainly cAMP-activated anion channels, the observed increase in Cl secretion is probably due to opening of Ca-activated basolateral K⁺ channels, hyperpolarization, and an increase in the driving force for Cl exit through CFTR. In CF tracheal gland cells, with CFTR defective, the ion transport response to MCh is virtually abolished (29), or at least diminished, according to a more recent report (8). Trout and coworkers (30) found that in porcine distal bronchi, the majority of acetylcholine-induced liquid secretion is dependent on Cl secretion, presumably from submucosal glands. In our experiments, the tracheal PD response to MCh was abolished and the mucus viscoelasticity was unchanged by pretreatment with the CFTR blocker NPPB. There was still a significant increase in fluid volume with MCh administration, although much less pronounced than without blocker pretreatment.

The observed changes in mucus viscoelasticity and tracheal PD with MCh and UTP are not consistent with a reflex mechanism, particularly given the modified responses obtained with DIDS or NPPB pretreatment. The changes are consistent with effects produced by these agents as the small volume of concentrated reagent diffuses into the tissue around the larynx. Although the dilution factor is uncertain, given the small volume delivered and the unknown tissue volume into which it must diffuse, the final concentrations in the trachea must be at least two orders of magnitude lower than the concentration administered, i.e., 10⁴ M or less. Also, we performed additional experiments to look at the time course of the inhibitory effect of DIDS instillation on tracheal PD, and found that it reached a stable level of 2 to 3 mV below control in about 10 min. As indicated earlier, care was taken not to allow the instillate to enter the trachea directly, which could have overwhelmed the limited volume of tracheal fluid present.

In this study, there was a small effect of DIDS on baseline PD, involving a mean change from 15.9 ± 1.6 to 13.5 ± 1.2 mV, as indicated in Figures 1A and 1B. This indicates that there is a certain portion of the baseline PD that is due to non-CFTR-dependent chloride secretion, because approximately 15 to 20% of the PD could be reduced by DIDS treatment. This suggests that in most C57BL/6 mice there is active alternative pathway chloride transport in baseline conditions. In addition, we observed that two of 16 C57BL/6 mice failed to exhibit a response to treatment with UTP. This may support the hypothesis that a subpopulation of C57BL/6 mice lack, or fail to activate, an alternative Cl conductance pathway. Kent and colleagues (31) speculated that the combination of a heritable absence of UTP-activated Cl channels with an experimentally engineered lack of CFTR could account for the development of lung disease. Thus, mice lacking in UTP responsiveness would be candidate progenitors for a more effective CFTR-knockout model of CF lung disease.

In summary, it was demonstrated that tracheal mucus hypersecretion owing to UTP in C57BL/6 mice is less vigorous than with MCh. These findings suggest that the population of Ca²⁺-dependent Cl channels stimulated by UTP-associated P₂ receptors in mice is limited compared with that of the mixed population of CFTR- and non-CFTR-dependent channels responding to secretagogues such as MCh.