Chloride channel function is linked to epithelium-dependent airway relaxation

Christopher N. Fortner, John N. Lorenz, and Richard J. Paul

Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously reported that substance P (SP) and ATP evoke transient, epithelium-dependent relaxation of mouse tracheal smooth muscle. Since both SP and ATP are known to evoke transepithelial Cl- secretion across epithelial monolayers, we tested the hypothesis that epithelium-dependent relaxation of mouse trachea depends on Cl- channel function. In perfused mouse tracheas, the responses to SP and ATP were both inhibited by the Cl- channel inhibitors diphenylamine-2-carboxylate and 5-nitro-2-(3-phenylpropylamino)benzoate. Relaxation to ATP or SP was unaffected by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), and relaxation to SP was unaffected by either DIDS or DNDS. Replacing Cl- in the buffer solutions with the impermeable anion gluconate on both sides of the trachea inhibited relaxation to SP or ATP. In contrast, increasing the gradient for Cl- secretion using Cl--free medium only in the tracheal lumen enhanced the relaxation to SP or ATP. We conclude that Cl- channel function is linked to receptor-mediated, epithelium-dependent relaxation. The finding that relaxation to SP was not blocked by DIDS suggested the involvement of a DIDS-insensitive Cl- channel, potentially the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. To test this hypothesis, we evaluated tracheas from CFTR-deficient mice and found that the peak relaxation to SP or ATP was not significantly different from those responses in wild-type littermates. This suggests that a DIDS-insensitive Cl- channel other than CFTR is active in the SP response. This work introduces a possible role for Cl- pathways in the modulation of airway smooth muscle function and may have implications for fundamental studies of airway function as well as therapeutic approaches to pulmonary disease.

smooth muscle; substance P; adenosine triphosphate; trachea; mouse


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE EPITHELIAL CELLS LINING the airways are the first surface exposed to inhaled compounds and may play a major role in determining the airway response to various stimuli. The epithelium may function as a source of relaxing factors or as a simple barrier against diffusion of inhaled agents (21). We have recently reported that substance P (SP) and ATP evoke transient, epithelium-dependent relaxations of constricted mouse trachea (10). These are likely to be physiologically relevant responses, since SP- immunoreactive fibers are present in mouse airways (28) and ATP is normally secreted into the airway lumen by the epithelium (15). Since both SP and ATP are also known to elicit transepithelial Cl- secretion in cultured epithelial monolayers (2, 24), it is possible that these two responses are functionally related. We thus tested the hypothesis that epithelium-dependent relaxation of mouse trachea depends on Cl- channel function. We used the mouse because it offers a powerful system in which the function of specific proteins may be investigated using gene-targeting strategies or transgenic approaches (22).

To study epithelial function in intact airways, we used an apparatus for evaluating constriction and relaxation responses in isolated, perfused mouse tracheas (10). This allowed side-specific delivery of inhibitors to either the airway lumen or smooth muscle. The effects of four Cl- channel inhibitors, DIDS, 4, 4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), diphenylamine-2-carboxylate (DPC), and 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), and Cl- substitution were studied in tracheas from wild-type mice. Based on our results in this part of the study and on the known sensitivity of cystic fibrosis transmembrane conductance regulator (CFTR) to these inhibitors (1, 11, 16), we further hypothesized that the observed relaxation to SP may be mediated through the CFTR Cl- channel. To test this second hypothesis, we used a genetic approach and studied tracheal function in a mouse line in which the CFTR channel has been deleted by gene targeting through homologous recombination [CFTR(-/-), purchased as cftrtm1Unc from The Jackson Laboratory, Bar Harbor, ME].

While Cl- currents appear to be involved in the response to osmotic stimuli (6), the present study offers the first evidence for the involvement of Cl- channels in receptor-mediated, epithelium-dependent modulation of airway smooth muscle contractility. This may have implications for fundamental studies of airway function as well as for therapeutic approaches to pulmonary disease.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tracheal tissue preparation. Mice aged 10-14 wk were selected for experimentation. Mice were killed by CO2 asphyxiation, and the trachea was removed and prepared as described below. Excised tracheas were maintained in physiological salt solution (PSS) of the following composition (in mM): 118 NaCl, 4.73 KCl, 1.2 MgSO4, 0.026 EDTA, 1.2 NaH2PO4, 2.5 CaCl2, and 11 glucose, buffered with 25 NaHCO3 to attain a pH of 7.4 at 37°C when bubbled with a mixture of 95% O2-5% CO2. For Cl- substitution studies, all Cl--containing salts were replaced with the same concentration of gluconate salts. When changing to Cl--free PSS, solutions were changed at least four times during the course of 1 h before the trachea was stimulated.

Perfused trachea preparations. The technique for perfusing an isolated mouse trachea with separate solutions bathing the smooth muscle and epithelium has been previously described in detail (10). Briefly, each trachea was tied with the ends around two cannulas. Differential pressure (Delta P) across the trachea was measured from wall taps near the cannula openings using pressure transducers (Cobe, Lakewood, CO) and recorded using the BioPac data acquisition system (BioPac Systems, Goleta, CA). At a perfusion rate of 16 ml/min, baseline Delta P was 1.10 ± 0.09 mmHg (n = 15) for tracheas without stimulation.

Force measurements in tracheal ring preparations. Tracheal rings were mounted isometrically to force transducers as previously described in detail (10). Paired tracheas from CFTR(-/-) and wild-type mice were mounted in the same chamber. Both epithelial and smooth muscle surfaces were bathed by the same buffer solution and thus exposed to identical concentrations of inhibitors or test agents.

Pharmaceuticals. Unless otherwise noted, chemicals were obtained from Sigma (St. Louis, MO). Acetylcholine chloride (ACh), SP, ATP, and DNDS (Molecular Probes, Eugene, OR) were dissolved in distilled water to produce concentrated stocks. DPC (Fluka, Milwaukee, WI), PGE2 (Cayman Chemical, Ann Arbor, MI), and indomethacin were dissolved in ethanol. DIDS and NPPB were dissolved in DMSO (Calbiochem, San Diego, CA). When added to the buffer solution, the final concentration of ethanol or DMSO was <1% vol/vol. In control experiments, this concentration of ethanol or DMSO alone had no effect on airway constriction or relaxation.

Data analysis. Data are expressed as means ± SE. The n value indicates the number of mice used. Standard ANOVA was used, with P < 0.05 taken as evidence of statistical significance. Concentration-response curves were fit with a logistic equation using Origin data analysis software (Microcal Software, Northampton, MA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Epithelium-dependent relaxation in perfused mouse trachea. In each experiment, control responses to luminal SP and ATP were measured in perfused tracheas constricted with 10 µM ACh in the extraluminal bathing solution (Fig. 1A). This concentration of ACh is approximately the ED80 for mouse trachea (10) and resulted in a repeatable constriction that increased Delta P by 1.19 ± 0.13 mmHg (n = 15) above baseline and was stable within 5 min of ACh addition. After tracheas were constricted, 10 nM SP was added to the luminal perfusate, and a transient relaxation was observed. The full magnitude of contraction was recovered within 15 min, at which time 10 µM ATP was added to the luminal perfusate and a second transient relaxation was observed. This was done without changing the luminal perfusate or the extraluminal bathing solution to maintain the same conditions and facilitate comparison of the SP and ATP responses.


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Fig. 1.   Reversible inhibition of relaxation to substance P (SP) and ATP by 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB). Typical experimental record from a mouse trachea contracted with 10 µM ACh in the extraluminal bath and then treated with 10 nM SP followed by 10 µM ATP in the luminal perfusate. A: control (untreated wild-type) trachea. B: record from the same experiment with the same trachea in the presence of 30 µM NPPB intraluminally. C: record from same trachea following 15-min washout after NPPB. AChe, contraction with extraluminal 10 µM ACh; We, extraluminal washout. Subscripts denote extraluminal (e) or intraluminal (i) administration of agents. Agents were added at the points indicated by the arrows and not removed until the points indicated by the "washout" arrows. Horizontal axis represents time; vertical axis represents differential pressure (Delta P).

Time control studies. Because our experimental design incorporated multiple measurements, we determined the reproducibility of mouse tracheal responses in tracheal rings from six mice. Ten cycles of contraction to ACh followed by treatment with SP or ATP and then subsequent treatment with the other relaxant (ATP or SP) were performed (Fig. 2). For the first five cycles and the final cycle, SP was given before ATP. For cycles 6-9, ATP was given before SP. Contraction to 10 µM ACh showed a decreasing trend over 10 h; however, the force was only significantly less for the contraction 8 h after dissection. The subsequent contraction was not different from any of the previous contractions. Relaxation to 10 nM SP depended on the time between treatments. This is not unexpected, since relaxation to SP is known to be tachyphylactic (10). With only 5 min between washout of the previous SP treatment and the subsequent contraction cycle, relaxation to SP was significantly reduced. Longer periods between SP exposures increased the relaxation to SP, such as occurred when ATP was given as the first relaxant and in the final cycle. While some variability in the SP and ATP responses was observed, there was no significant loss of relaxation to SP or ATP over the 10-h time control, with the exception of the second SP treatment following the shorter (5-min) washout period.


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Fig. 2.   Time controls for mouse tracheal constriction and epithelium-dependent relaxation. Values are averages ± SE of isometric force from n = 6 tracheas tested over the course of 10 h, with 10 repeated cycles of constriction with 10 µM ACh followed by either 10 nM SP or 10 µM ATP and then the other relaxant (SP or ATP). Constriction and relaxation responses were repeatable for up to 8 h, at which point the force of constriction was slightly reduced compared with the initial cycle.

Effects of Cl- channel inhibitors on epithelium-dependent relaxation. After the control measurements and subsequent 10-min washout period, inhibitors were added to the luminal perfusate or extraluminal bath as specified. DIDS, DNDS, and DPC were added 15 min and NPPB 20 min before the trachea was constricted and remained present for the duration of the experiment. The maximum concentrations of DIDS (1 mM) and DPC (2.5 mM) were chosen based on reported concentrations that effectively inhibit Cl- channels (5, 14, 17, 26, 30). NPPB (10 µM) blocked relaxation of tracheal rings to SP and ATP (n = 7). Ten micromolar NPPB was less effective at blocking the relaxation responses in perfused tracheas; thus 30 µM was used (Fig. 1B). The relaxation to ATP was blocked by DIDS, DPC, and NPPB but not by DNDS (tracheal rings constricted with 10 µM ACh relaxed 69 ± 4 vs. 54 ± 3% in controls, n = 6). NPPB inhibited relaxation to ATP more effectively when delivered to the luminal perfusate than when administered to the outer bathing solution only (Fig. 3A). Inhibition by DPC or NPPB was reversible on replacement of the PSS. DIDS inhibition of ATP relaxation was only partially reversible, even following a 1-h recovery period. In contrast to the ATP response, the relaxation to SP was unaffected by DIDS or DNDS (62 ± 3 vs. 71 ± 4% in controls, n = 6) but was blocked by DPC and NPPB. As with the ATP response, 30 µM NPPB in the lumen blocked the relaxation to SP more effectively than extraluminal NPPB (Fig. 3B).


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Fig. 3.   Effect of Cl- channel inhibitors on epithelium-dependent relaxation. Inhibitors were added 15-20 min before constriction. Peak relaxation is expressed as a percentage of the constriction response to 10 µM ACh. All inhibitors were added to the luminal perfusate except the group treated with NPPB extraluminally (NPPB EL). A: average values ± SE for tracheas constricted with extraluminal ACh followed by luminal treatment with 10 µM ATP. Both DIDS and diphenylamine-2-carboxylate (DPC) significantly reduced relaxation compared with control. B: average values ± SE for tracheas constricted with extraluminal ACh followed by luminal treatment with 10 nM SP. DPC significantly reduced relaxation compared with control (n = 9 for controls, n = 4 for each concentration of inhibitor).

Effects of Cl- substitution in perfused tracheas. An additional method for inhibiting Cl- currents is to replace Cl- with an impermeable anion such as gluconate (11). To obtain completely Cl--free conditions, potassium gluconate rather than ACh was used to depolarize and constrict the smooth muscle. In Cl--free PSS, the constriction to 40 mM potassium gluconate was <50% of control on average. In control experiments (n = 3), we demonstrated that both SP and ATP could relax tracheas constricted with 40 mM potassium gluconate (Fig. 4A). Figure 4B shows the typical inhibitory effects of Cl--free PSS on the relaxation to SP or ATP. Cl--free PSS significantly inhibited the relaxation to SP (24 ± 12% of constriction vs. 61 ± 8% in controls) or ATP (20 ± 7% of constriction vs. 60 ± 9% in controls). Consistent with the results obtained with Cl- channel inhibitors, the studies in Cl--free PSS suggest a linkage between Cl- currents and epithelium-dependent relaxation.


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Fig. 4.   Effect of Cl- substitution on epithelium-dependent relaxation. Shown is a typical experimental record (n = 4) from a mouse trachea contracted with 40 mM potassium gluconate in the extraluminal bath and then treated with 10 nM SP followed by 10 µM ATP in the luminal perfusate. A: control (untreated wild-type) trachea. B: experimental record from the same experiment with the same trachea in Cl--free buffer. Ke, contraction with extraluminal 40 mM potassium gluconate. Subscripts denote extraluminal (e) or intraluminal (i) administration of agents. Agents were added at the points indicated by the arrows and not removed until the points indicated by the washout arrows. Horizontal axis represents time; vertical axis represents differential pressure (Delta P).

To determine whether the reduction in contraction in Cl--free PSS was due to increased production of relaxing prostanoids, the effect of Cl--free PSS on contractions to ACh was studied in tracheal rings treated with indomethacin. Isometric force in response to 10 µM ACh in Cl--free PSS was reduced to 74 ± 6% of the force observed in regular PSS (n = 6). Ten micromolar indomethacin was added to the tissue bath 20 min before contraction with 10 µM ACh and was present for the duration of the experiment. In the presence of indomethacin, Cl--free PSS reduced the contraction to ACh to 65 ± 3% of the force observed in regular PSS (n = 6), not significantly different from controls (P = 0.17). The effects of Cl- substitution were reversible; after airways were rinsed with PSS to restore Cl- to the system, subsequent contractions regained their full magnitude. Thus the reduced constriction in Cl--free PSS was unlikely to be due to increased production of a prostanoid relaxing factor.

Epithelium-dependent relaxation with Cl--free PSS in the lumen only. If Cl- channel currents are functionally linked to the epithelium-dependent relaxation to SP or ATP, one possible way to enhance the responses would be to increase the gradient for Cl- secretion using Cl--free PSS in the airway lumen only. We also used submaximal concentrations of SP or ATP to avoid potential saturation of the responses. Constriction to 10 µM ACh was not affected when Cl--free PSS was perfused through the lumen (Delta P = 0.90 ± 0.10 mmHg with luminal Cl--free PSS vs. 0.95 ± 0.10 mmHg with regular PSS; n = 8). As shown in Fig. 5, Cl--free PSS only in the lumen enhanced relaxation to 1 nM intraluminal SP or 0.3 µM intraluminal ATP. Relaxation to SP was increased by 31% above control (relaxation with luminal Cl--free PSS was 60 ± 8 vs. 46 ± 10% in control, P < 0.05; n = 8), and relaxation to ATP was increased by 43% (73 ± 3 vs. 51 ± 8% in control, P < 0.02; n = 7).


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Fig. 5.   Relaxation to SP or ATP with Cl--free physiological salt solution (PSS) in lumen only. Shown is a typical experimental record (n = 7-8) from a mouse trachea perfused luminally with Cl--free PSS. Relaxation to 1 nM SP or 0.3 µM ATP was enhanced. A: control. B: Cl--free PSS in lumen only. AChe, contraction with extraluminal 10 µM ACh. Subscripts denote extraluminal (e) or intraluminal (i) administration of agents. Agents were added at the points indicated by the arrows. Horizontal axis represents time; vertical axis represents differential pressure (Delta P).

PGE2 relaxation. The inhibition of these epithelium-dependent relaxations by interventions designed to affect Cl- currents could also be attributed to direct effects on the ability of the smooth muscle to relax. To provide a positive control for relaxation when responses to both SP and ATP were blocked, constricted tracheas were treated with PGE2. Smooth muscle-specific relaxation was elicited by addition of PGE2 only to the extraluminal bath. The concentrations of PGE2 were chosen because they produced a relaxation similar to that of SP or ATP. Perfusing the lumen with DPC had a small but statistically significant effect on relaxation to 30 nM PGE2 and no effect on relaxation to 100 nM PGE2. Ten micromolar NPPB slightly decreased relaxation to PGE2 in tracheal rings (Fig. 6), and this effect was not reversible on washout of NPPB, in contrast to relaxation to SP or ATP. While the Cl- channel inhibitors nearly abolished the epithelium-dependent responses to SP or ATP (reducing them to 2-26% of control), the smooth muscle relaxation to PGE2 was only moderately affected (73-93% of control). Cl--free PSS did not affect the sensitivity of the concentration response to PGE2 (IC50 = 104 ± 67 nM compared with 15 ± 1 nM in control) but marginally reduced the maximum relaxation (84% of relaxation was retained compared with control). Thus the effects of Cl--free PSS and the various Cl- channel inhibitors do not exert their major effects at the smooth muscle level.


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Fig. 6.   Effect of Cl- channel inhibitors on relaxation to PGE2. Values are averages ± SE for relaxation to PGE2 expressed as a percentage of constriction to 10 µM ACh. Isometric force in tracheal rings was used to assess the effects of NPPB (n = 6). Results for DPC were collected by perfusing the trachea with 2.5 mM DPC and adding PGE2 to the extraluminal bath (n = 4). *Small but statistically significant (P < 0.05) reductions.

Isometric force measurements in tracheal rings from CFTR-deficient mice. The CFTR Cl- channel is blocked by DPC but is unaffected by DIDS (1). Our observation that the relaxation to SP was also blocked by DPC but not by DIDS suggested the potential involvement of the CFTR Cl- channel. Tracheas from CFTR(-/-) mice were paired with wild type and were mounted in the same bath for isometric force measurements. There were no differences between CFTR(-/-) and wild-type tracheas in gross morphological properties (Table 1) or constriction to ACh (Table 2). After constriction with 10 µM ACh, tracheal pairs were treated with SP or ATP. In wild-type trachea, DIDS inhibited the relaxation to ATP but not that to SP (Fig. 7), in agreement with the studies on the perfused trachea. NPPB (10 µM) blocked the relaxation both to SP and to ATP in a reversible manner. The relaxations to SP and ATP persisted in the tracheas from CFTR(-/-) mice, and the magnitude of relaxation was not significantly different from that in the wild type (Fig. 7). It was not possible to study the effects of DPC in the tracheal ring preparation because 2.5 mM DPC completely blocked contraction to ACh (n = 4). As with DPC blockade of the SP or ATP relaxations, the blockade of contraction was reversible. Normal contractions to ACh were observed 30 min after DPC washout.

                              
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Table 1.   Tracheal properties for CFTR(-/-) and wild-type mice


                              
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Table 2.   Effects of Cl- channel blockade on ACh (10 µM) contractions of mouse tracheas



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Fig. 7.   Relaxation to SP or ATP in tracheas from cystic fibrosis transmembrane conductance regulator-deficient CFTR(-/-) mice. Paired tracheal rings from wild-type and CFTR(-/-) mice were studied in an isometric force measurement apparatus. Peak relaxation to SP or ATP is expressed as a percentage of the constriction response to 10 µM ACh. Bars are means ± SE for n = 3 CFTR(-/-) mice treated with NPPB; n = 7 for all other groups. A: relaxation to 10 µM ATP. Both DIDS and NPPB significantly reduced relaxation compared with control. B: relaxation to 10 nM SP. NPPB significantly reduced relaxation compared with control. There was no significant difference in the responses of CFTR(-/-) tracheas under control conditions, and there was no difference in the effects of Cl- channel inhibitors compared with wild-type tracheas.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The role of the airway epithelium in modifying the contractility of the underlying smooth muscle is not fully understood. It has been suggested that the primary function of the epithelium is to provide a barrier of protection between the airway smooth muscle and inhaled irritants (21). More recently, studies have demonstrated that the epithelium can be an active source of mediators that relax constricted airways (7, 10, 27). SP and ATP are two agonists that stimulate an epithelium-dependent relaxation of constricted airways (10). Both of these agonists have also been shown to stimulate Cl- secretion across the airway epithelium into the airway lumen. The first objective of this study was to investigate whether these two actions were functionally linked. Specifically, is Cl- channel activity necessary for SP or ATP to elicit epithelium-dependent relaxation of airways?

To test the hypothesis that the epithelium-dependent relaxation to SP and ATP depends on Cl- channel activity, we evaluated the effects of various pharmacological inhibitors of Cl- channels on SP and ATP responses. The concentrations of Cl- channel inhibitors were chosen based on published values shown to effectively block Cl- channels (5, 11, 14, 17, 26, 30). Although the specificity of Cl- channel inhibitors is not absolute, the combined evidence from our studies with several different inhibitors or Cl- substitution supports a dependence on Cl- currents for receptor-mediated, epithelium-dependent relaxation.

Of the inhibitors tested, NPPB is the most potent Cl- channel inhibitor (29) and is believed to be the most selective for Cl- channels. To investigate whether epithelial or smooth muscle Cl- currents are more important in the relaxation to SP or ATP, we used side-specific delivery of NPPB to either the airway lumen or outer perfusate in our perfused trachea system. We first used 10 µM NPPB, since this concentration effectively blocked relaxation of tracheal rings to SP and ATP. Interestingly, when delivered intraluminally, extraluminally, or bilaterally, 10 µM NPPB was less effective at blocking the relaxation to SP or ATP in the perfused trachea than it was in tracheal rings. Bilateral treatment with NPPB should be equivalent to the conditions in the isometric force bath, with the exception of constant flow through the airway lumen. Thus it appears that the effectiveness of NPPB blockade may depend on flow across the epithelium. At 30 µM, intraluminal or extraluminal NPPB inhibited relaxation to SP or ATP; however, the inhibitor was greater than twofold more effective (Fig. 2) when delivered luminally. This finding suggests that blockade of epithelial Cl- channels is more important than blockade of smooth muscle Cl- channels for inhibiting epithelium-dependent relaxation to SP or ATP.

We also used nominally Cl--free solutions in which Cl- has been replaced with an anion that does not pass easily through Cl- channels to test whether the SP and ATP responses depend on Cl- currents. Such techniques have been successfully used in experiments on cultured epithelial cells; however, the use of intact trachea in our experimental system makes interpretation of these data more complex. To completely remove Cl- in the PSS that superfuses the smooth muscle in some experiments, we changed the constricting stimulus to potassium gluconate, since ACh was typically used to constrict the trachea in PSS. Smooth muscle contraction was reduced in Cl--free PSS. Cl- is an important intracellular anion in smooth muscle, and removing Cl- from the system may directly impair smooth muscle contraction and/or relaxation.

The reduced contractions in the absence of Cl- might be due to stimulation of the release of a relaxing prostanoid. Contractions to ACh were reduced in Cl--free PSS, a finding similar to that reported for rat tracheal smooth muscle (25). Indomethacin did not prevent the reduction in airway contractility in Cl--free PSS, thereby excluding eicosanoid release as a mechanism to explain the effects of Cl--free PSS on airway constriction. Because Cl--free PSS reduced contraction (Table 2), direct comparison of its blockade of SP and ATP to their effects in control PSS is not possible. However, since lower levels of constriction are generally associated with greater effectiveness of relaxing agents, the finding that Cl- substitution inhibits rather than augments the relaxation to SP or ATP further supports the conclusion that epithelium-dependent relaxation depends on Cl- channel activity.

The effect of DIDS on epithelium-dependent relaxation to ATP may be due to DIDS blockade of purinoceptors (4). DNDS is similar to DIDS in its effects on Cl- channels (3) yet does not block purinoceptors (20). We found that DNDS did not block relaxation to either SP or ATP. Regardless of the effects of DIDS on ATP-induced relaxation, the important finding from the DIDS experiments is that the response to SP is not sensitive to DIDS inhibition.

It is possible that the interventions used to inhibit Cl- channels blocked relaxation at the smooth muscle level. For the studies using DIDS, relaxation to SP provided a valuable internal positive control for relaxation, since SP relaxation was unaffected in the presence of DIDS. In the cases where relaxation to both SP and ATP was blocked, we investigated the effects of these inhibitors on relaxation to exogenous PGE2. PGE2 was chosen as a positive control since relaxation to SP or ATP is likely to involve an eicosanoid (10), and PGE2 is a possible mediator of SP relaxation (23). In tracheas where relaxation to both SP and ATP were blocked by Cl- channel inhibitors or Cl--free PSS, 100 nM PGE2 relaxed constricted tracheas similar to the control responses to SP or ATP. One interesting finding was that NPPB reduced the response to PGE2, although not to the extent that relaxation to SP or ATP was blocked. This effect on relaxation to PGE2 did not reverse on removal of NPPB; however, relaxation to SP or ATP recovered in these same tracheas after NPPB washout. This suggests that the irreversible inhibition of NPPB on relaxation to PGE2 was different from its effects on relaxation to SP or ATP. For relaxation to PGE2 in Cl--free PSS, the shift in sensitivity may be due to alterations in smooth muscle pH. Surrounding smooth muscle with gluconate-substituted PSS has been reported to raise intracellular pH (12), and alkalinization of smooth muscle shifts the sensitivity to certain relaxation stimuli (18).

One Cl- channel that has been characterized based on its sensitivity to DPC while being insensitive to DIDS is the CFTR Cl- channel (1). Our results suggested that the epithelium-dependent relaxation to SP may be mediated through the CFTR channel. We tested this hypothesis using mice deficient in CFTR (19). These mice develop an intestinal pathology similar to that of the human disease cystic fibrosis, but there is no overt phenotype reported in the lungs of CFTR(-/-) mice (8). Measurements of isometric force were used to compare tracheal rings from CFTR(-/-) and wild-type mice in the same PSS and exposed to identical concentrations of test agents. There were no differences in the ACh concentration-force relationship for CFTR(-/-) and wild-type tracheas. The finding that SP-induced relaxation was intact in the CFTR(-/-) trachea indicated that CFTR function is not required for airway relaxation to SP. The SP relaxation may be mediated through other Cl- channels based on the documented persistence of DIDS-insensitive Cl- currents in the trachea from CFTR(-/-) mice (9).

The exact mechanism by which epithelium-dependent relaxation involves Cl- channels is not known. We have previously reported that indomethacin irreversibly blocks the relaxation to SP and ATP (10). It is presumed that this effect is due to blockade of cyclooxygenase-dependent production of an eicosanoid relaxing factor. Recent studies have also suggested a possible role for Cl- currents in epithelium-dependent relaxation to osmotic stimuli (6); however, the airway response to osmotic stimuli is not inhibited by indomethacin, unlike our observed responses to SP and ATP (10). We found no reports of indomethacin, DPC, or NPPB interfering directly with the receptors for SP or ATP.

Although the specific mechanism by which Cl- movement facilitates epithelium-dependent airway relaxation remains to be elucidated, the current study offers the first evidence for a role of Cl- currents in epithelium-mediated relaxation of mouse airways. Since both the epithelial and smooth muscle components may be affected by the experiments demonstrating the dependence on Cl- currents, one cannot unambiguously assign a specific cellular locus for the effect. However, some of the observed results suggest that epithelial Cl- channels are of greater importance in the epithelium-dependent relaxation to SP or ATP. First, the relatively impermeable Cl- channel inhibitor NPPB was more effective when delivered luminally compared with when superfused on the smooth muscle. Additionally, the finding that DPC blocks constriction of tracheal rings combined with the fact that DPC in the lumen only did not affect constriction in perfused trachea suggests that little or no DPC reached the smooth muscle, and thus the major site of its effects was most likely the epithelium. Finally, the strongest evidence to support a dependence on epithelial Cl- channels was the finding that Cl--free PSS in only the lumen enhanced the relaxation to SP or ATP. Under these conditions, the smooth muscle was not exposed to Cl--free conditions; however, the Cl- gradient for epithelial Cl- secretion would be increased. This did not appear to alter any basal release of mediators from the epithelium, since constriction to ACh was not affected by luminal Cl--free PSS. Based on the combined evidence from these three experiments, it is likely that our results reflect a greater influence of epithelial Cl- currents.

This involvement of Cl- channels in receptor-mediated, epithelium-dependent relaxation of the airways may be of value in the therapy of airway obstructive diseases such as asthma. Pharmaceuticals may be developed to stimulate Cl- currents and thus enhance the release of endogenous epithelium-derived relaxing factors. The exact pathogenesis of cystic fibrosis also remains unclear, and it has been suggested that there is underlying hyperfunction of airway smooth muscle in cystic fibrosis (13). Perhaps in humans where there is insufficient compensation for the loss of the CFTR channel, impaired release of an epithelium-dependent airway-relaxing factor may contribute to the pathogenesis of cystic fibrosis. The present study may therefore have implications for the therapy of one disease and for better understanding of another.


    ACKNOWLEDGEMENTS

This work was supported by an MD/PhD Scholar Award (to C. N. Fortner) and National Heart, Lung, and Blood Institute Grants HL-61974 (to R. J. Paul) and HL-54829 (to R. J. Paul).


    FOOTNOTES

Address for reprint requests and other correspondence: C. N. Fortner, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0576 (E-mail: chris.fortner{at}uc.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 4 January 2000; accepted in final form 7 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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