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Published ahead of print on August 28, 2008, doi:10.1165/rcmb.2006-0444OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 40, pp. 217-222, 2009
© 2009 American Thoracic Society
DOI: 10.1165/rcmb.2006-0444OC

Role of the Cystic Fibrosis Transmembrane Conductance Channel in Human Airway Smooth Muscle

Marie-Claire Michoud1, Renaud Robert2, Muhannad Hassan1, Barry Moynihan1, Christina Haston1, Vasanthi Govindaraju1, Pasquale Ferraro3, John W. Hanrahan2 and James G. Martin1

1 Seymour Heisler Laboratory of the Montreal Chest Institute and Meakins-Christie Laboratories, McGill University; 2 Physiology Department, McGill University, Montreal, Quebec, Canada; and 3 University of Montreal Hospital Center, Montreal, Quebec, Canada

Correspondence and requests for reprints should be addressed to James G. Martin, M.D., D.Sc., Meakins Christie Laboratories, Department of Medicine, McGill University, 3626 St. Urbain, Montreal, PQ, H2X 2P2 Canada. E-mail: james.martin{at}mcgill.ca


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 Statistical Analysis
 RESULTS
 DISCUSSION
 References
 
Patients with cystic fibrosis (CF) suffer from asthma-like symptoms and gastrointestinal cramps, attributed to a mutation in the CF transmembrane conductance regulator (CFTR) gene present in a variety of cells. Pulmonary manifestations of the disease include the production of thickened mucus and symptoms of asthma, such as cough and wheezing. A possible alteration in airway smooth muscle (ASM) cell function of patients with CF has not been investigated. The aim of this study was to determine whether the (CFTR) channel is present and affects function of human ASM cells. Cell cultures were obtained from the main or lobar bronchi of patients with and without CF, and the presence of the CFTR channel detected by immunofluorescence. Cytosolic Ca2+ was measured using Fura-2 and dual-wavelength microfluorimetry. The results show that CFTR is expressed in airway bronchial tissue and in cultured ASM cells. Peak Ca2+ release in response to histamine was significantly decreased in CF cells compared with non-CF ASM cells (357 ± 53 nM versus 558 ± 20 nM; P < 0.001). The CFTR pharmacological blockers, glibenclamide and N-phenyl anthranilic acid, significantly reduced histamine-induced Ca2+ release in non-CF cells, and similar results were obtained when CFTR expression was varied using antisense oligonucleotides. In conclusion, these data show that the CFTR channel is present in ASM cells, and that it modulates the release of Ca2+ in response to contractile agents. In patients with CF, a dysfunctional CFTR channel could contribute to the asthma diathesis and gastrointestinal problems experienced by these patients.

Key Words: cystic fibrosis • histamine • chloride channel • glibenclamide • oligodeoxynucleotide



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 Statistical Analysis
 RESULTS
 DISCUSSION
 References
 
This study shows that the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) channel is involved in the regulation of intracellular calcium in airway smooth muscle cells. Mutations in the CFTR channel may contribute to the asthma diathesis observed in patients with CF.

 
A substantial fraction of patients with cystic fibrosis (CF) has symptoms of asthma, such as cough and wheezing, and demonstrates airway hyperresponsiveness to histamine and/or methacholine (1, 2). Although these manifestations may be partly due to epithelial abnormalities, there is a growing consensus that airway hyperresponsiveness mainly reflects airway smooth muscle (ASM) dysfunction (3). Indeed, ASM cells from patients with asthma have been studied and shown to differ from individuals without asthma (4). However, the ASM cells from patients with CF have not, to our knowledge, been investigated. In addition, although consequences of a loss of function of the CF transmembrane conductance regulator (CFTR) channel have been investigated extensively in epithelial cells, little is known about the physiological role of this channel in other cell types.

The primary function of the CFTR channel is to mediate cAMP-activated Cl– conductance across the plasma membrane of epithelial cells. Consistent with this Cl– channel function, mutations in the CFTR gene lead to clinical manifestations, such as the thick mucus secretion that impairs airway clearance and obstructs ducts in the pancreas and other organs. Furthermore, additional functions have been attributed to the CFTR, including regulation of the epithelial Na+ channel and other channels and transporters (5–11). Effects of the CFTR channel on vesicle trafficking, bicarbonate transport, and the expression of inflammatory mediators, such as regulated upon activation, normal T-cell expressed and secreted (RANTES), IL-8, IL-10, and inducible nitrous oxide synthase, have also been reported (12, 13).

Studies of CFTR expression indicate that it is also present in nonepithelial cell types, including lymphocytes, cardiac ventricular cells, endothelial cells, and, more recently, in rodent vascular and tracheal smooth muscle cells (14–18). Its expression in cardiac myocytes was described 10 years ago (19, 20) and, although there is some evidence for alternative splicing at exon 5, electrophysiological studies indicate that the properties of cardiac CFTR are closely similar, if not identical, to those of epithelial CFTR (21). However, the physiological function of the channel, and its possible role in modulating the contractile properties of cardiac myocytes, remain uncertain. Very recently, CFTR has been reported to modulate rodent vascular and tracheal smooth muscle relaxant responses (16–18). In precontracted arteries of rat, or tracheas of Cftr(+/+) mice, vasoactive intestinal peptide (VIP) and other CFTR activators induced vasorelaxation. Conversely, the absence of CFTR in the vascular smooth muscle cells was associated with a higher contraction in Cftr(–/–) mice, suggesting that loss or dysfunction of CFTR impairs the physiological control of vascular tone. In the present study, we investigate the possible role of CFTR Cl– channel to modulate human ASM contractility and relaxation by determining if it is present in human ASM, and whether it influences intracellular Ca2+ homeostasis, a critical second messenger in smooth muscle contraction.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
Statistical Analysis
 RESULTS
 DISCUSSION
 References
 
Cell Cultures
Fragments of lobar bronchi were obtained from donors and recipients of lung transplants at the time of transplantation. The ASM cells from subjects without CF were isolated as previously described (22). Briefly, the tissue was washed thoroughly in Ca2+-free Hanks' balanced salt solution (HBSS), cut into 5-mm x 5-mm pieces, and digested for 90 minutes at 37°C in HBSS containing 0.4 mg/ml collagenase type IV, 1 mg/ml soybean trypsin inhibitor, and 0.38 mg/ml elastase type IV. The resulting cell suspension was filtered through 125-µm Nytex mesh, and dissociated cells collected by centrifugation and then resuspended in 1:1 Dulbecco's modified Eagles medium/Ham's F12 medium supplemented with 10% FBS, 0.244% NaHCO3, penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin (0.25 µg/ml), and plated in 25-cm2 culture flasks.

ASM cells from subjects with CF (five females and two males, all affected by the {Delta}F508 mutation) were isolated and cultured using a modification of the technique described by Randell and colleagues (23) to avoid contamination. Namely, the tissue was cut into small rectangles of 10 mm x 5 mm and soaked for 20 minutes in cold Hanks' buffer containing 0.5 mg/ml dithiothreitol and 10 µl/ml of DNase type I to remove mucus, debris, and to decrease the bacterial burden. The fragments were then cut into smaller pieces and placed in a cell dissociation medium made of HBSS, to which 0.4 mg/ml collagenase type IV, 1 mg/ml soybean trypsin inhibitor, and 0.38 mg/ml elastase type IV, penicillin (100 U/ml), streptomycin (100 µg/ml), ceftazidime (100 µl/ml), ciprofloxacin (20 µl/ml), colistin (5 µg/ml), tobramycin (80 µg/ml), and gentamycin (50 µg/ml) were added. The tissue was digested for 90 minutes at 37°C, and the resulting cell suspension filtered and plated as described above. The same antibiotics were added to the culture medium for 48–72 hours, after which the cells were exposed to the same culture medium as the non-ASM CF cells.

Confluent cells were detached with a 0.025% trypsin and 0.02% EDTA solution and grown on 25-mm-diameter glass coverslips for Ca2+ imaging and immunofluorescence studies. Confluent cells from first to fourth passage were used. ASM cells in primary cultures were identified by immunostaining for smooth muscle cell–specific {alpha}-actin and calponin (and Western blotting for myosin light chain kinase and calponin).

The project was approved by the local institutional review boards.

Immunofluorescence Studies
After fixation and permeabilization, the cells were incubated for 60 minutes with CFTR antibody (monoclonal mouse anti-human CFTR, C terminus antibody, clone 24-1 [R&D systems]) (23) or calponin antibody (monoclonal anti-calponin antibody, C2687; Sigma-Aldrich, St. Louis, MO) in PBS containing 1% BSA followed by incubation with an FITC-conjugated secondary antibody (Alexa Fluor 488 goat anti-mouse IgG2a; Molecular Probes, Eugene, OR). In control experiments, primary antibody was omitted and the cells incubated in PBS/BSA alone or in the presence of concentration-matched isotype antibody IgG1 (24).

In tissue sections, staining was preceded by high-temperature epitope unmasking in antigen retrieval solution (Vector Laboratories, Burlington, ON, Canada) and permeabilization in 0.2% Triton X-100 (Sigma-Aldrich). Sections were then blocked with 20% horse serum (Vector Laboratories) in universal blocking solution (Dako Cytomation, Mississauga, ON, Canada), and the anti-CFTR C-terminal monoclonal primary antibody was detected with biotinylated horse anti-mouse IgG, avidin/biotin-alkaline phosphatase complex, and then developed with BCIP/NBT chromogen substrate (Vector Laboratories). Double immunostaining with anti–smooth muscle {alpha}-actin monoclonal antibody was done after that in a similar way, but Vector Red was used for developing, and methyl green (Sigma-Aldrich) was used as a counterstain.

Ca2+ Imaging
Cells on 25-mm-diameter slides were loaded with the Ca2+-sensitive fluorescent dye Fura-2AM (Molecular Probes), as previously described (25), and imaged using an intensified charge-coupled device camera (IC200) and PTI software (Photon Technology International, Princeton, NJ) at a single emission wavelength (510 nm) with a double excitatory wavelength (340 and 380 nm). The fluorescence ratio (345:380) was measured in individual cells, and the intracellular free Ca2+ ([Ca2+]i) calculated assuming a Ca2+ dissociation constant for Fura-2 of 224 nM. Maximum ratio was determined in cells exposed to 10–5 M ionomycin in the presence of 1.3 mM CaCl2 and minimum ratio in Ca2+-free Hanks' buffer to which EGTA (10–3 M) and ionomycin (10–5 M) had been added. Background fluorescence was automatically subtracted.

Antisense experiments.
Antisense experiments: 5'- and 3'-end phosphorothioate-modified antisense oligodeoxynucleotide (ODN): 5'-TTT TCC AGA GGC GAC CTC TGC AT-3', and sense ODN 5'-ATG CAG AGG TCG CCT CTG GAA AA-3', which encompass the first 23 base pairs of the CFTR transcript beginning at the translation initiation, AUG (26), were added to cell cultures at a concentration of 0.05–1.00 µM. Cells were transfected using FuGENE 6 (Roche) as described by Peng and colleagues (27). Briefly, a transfection cocktail ratio of 2.5 ODN (0.05–1 µM) to 1 FuGENE 6 was added to growth-arrested subconfluent cells (60–70% confluence) in serum-free medium supplemented with BSA (1 mg/ml), transferrin (5 µg/ml), and insulin (5.7 µg/ml). The cells were incubated with growth medium containing FuGENE 6 alone (no ODN). The cells were incubated for 72 hours, after which the medium was removed and the cells prepared for Ca2+ imaging experiments or immunocytochemistry.


   Statistical Analysis
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 Statistical Analysis
RESULTS
 DISCUSSION
 References
 
Data are presented as means ± SEM. The Student's t test was used to compare means, and a difference was considered to be statistically significant when the P value was less than 0.05.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 Statistical Analysis
 RESULTS
DISCUSSION
 References
 
Smooth Muscle Cell Phenotype and CFTR Expression
Both CF- and non-CF–cultured ASM cells expressed smooth muscle–specific {alpha}-actin. In addition, the cell phenotype was confirmed by immunostaining with calponin antibody (24), as shown in Figure 1.


Figure 1
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  		Figure 1. Expression of calponin in non–cystic fibrosis (CF) (A) and CF (B) airway smooth muscle (ASM) cells in culture. Scale bars = 50 µm.

 
CFTR expression was assessed in airway tissue and cultured non-CF ASM cells by immunostaining using a monoclonal antibody against the CFTR carboxyl terminus. Figure 2A is a tissue section from a lobar bronchus of a subject without CF. Double immunostaining shows that CFTR (dark blue) is present in mucus glands and in the smooth muscle layer (stained red). Figure 2B shows diffuse expression of CFTR throughout the cytoplasm of cultured non-CF ASM cells.


Figure 2
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  		Figure 2. Expression of CF transmembrane conductance regulator (CFTR) in ASM cells. (A) A tissue section of a lobar bronchus of a subject without CF. Double immunostaining shows that CFTR (dark blue) is present in mucus glands and in the smooth muscle layer (stained red). (B) Diffuse expression of CFTR throughout the cytoplasm of cultured non-CF ASM cells. Scale bars = 50 µm.

 
Effects of Histamine on Intracellular Ca2+ Release
The effect of a contractile agonist, histamine, on Ca2+ release was measured in cells isolated from both subjects with and without CF. As shown in Figure 3A and 3B, exposure to 10–4 M histamine induced the release of Ca2+ in both CF and non-CF cells, but the peak release was lower in CF ASM cells compared with non-CF cells (357 ± 53 nM [n = 56 cells, 7 slides] versus 558 ± 20 nM [n = 134 cells, 16 slides] P < 0.001) (Figure 3B). Resting [Ca2+]i was slightly but significantly lower in CF than in non-CF cells (107 ± 4 nM versus 129 ± 6 nM; P = 0.001). Considering that the cultured CF smooth muscle cells had been exposed to a mixture of antibiotics for the first 48–72 hours, we conducted experiments to ascertain that this decreased response to histamine by the CF cells was not secondary to antibiotic exposure. The results show that exposure to antibiotics for 48–72 hours did not affect Ca2+ release in non-CF ASM cells. Peak [Ca2+]i response to 10–4 M histamine was 537 ± 24 nM (n = 47 cells, 6 slides) in antibiotic-exposed cells, while peak response of cells exposed to the standard growth medium was 532 ± 23 nM (n = 48 cells, 6 slides).


Figure 3
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  		Figure 3. Histamine-induced Ca2+ release in non-CF and CF ASM cells. (A) A representative example of Ca2+ release induced by histamine (100 µM) in a non-CF and a CF ASM cell. Arrows indicate the time at which histamine was added to the extracellular fluid. (B and C) The average resting (R) intracellular Ca2+concentrations ([Ca2+]i) and peak Ca2+ releases in response to histamine (100 µM) in CF (hatched bars) and non-CF (open bars) ASM cells in Ca2+-rich (B) and Ca2+-free (C) medium. Data are shown as means + SEM. *P < 0.001; **P = 0.001.

 
The role of extracellular Ca2+ was tested by measuring Ca2+ mobilization after histamine stimulation when cells were placed in Ca2+-free medium. The increase in Ca2+ was 242 ± 15 nM (n = 90 cells, 11 independent measurements) in non-CF cells versus 268 ± 45 nM (n = 58 cells, 8 independent measurements) in CF cells (Figure 3C). These results show that the difference in peak Ca2+ release in response to histamine between CF and non-CF ASM cells was abolished, and that differences are due to extracellular Ca2+ influx, probably through ion channels.

Effects of CFTR Channel Blockers on Ca2+ Release
The role of CFTR channel activity in regulating intracellular Ca2+ homeostasis was investigated further by exposing non-CF ASM cells to the well established CFTR channel inhibitors, glibenclamide and N-phenylanthranilic acid (DPC). Following incubation of non-CF ASM cells for 20 minutes with 100 µM glibenclamide, peak [Ca2+]i in response to 10–4 M histamine was significantly decreased compared with vehicle-treated cells (227 ± 18 nM [n = 67 cells, 8 slides] versus 416 ± 37 nM [n = 72 cells, 9 slides]; P < 0.001) (Figure 4A). Mean resting [Ca2+]i was slightly elevated in the presence of glibenclamide; however, this difference did not quite reach statistical significance (113 ± 8 nM versus 92 ± 8 nM in vehicle-treated cells; P = 0.085) (Figure 4A). Exposure to DPC (100 µM) also had a significant effect on Ca2+ release induced by histamine: peak Ca2+ was 206 ± 31 nM (n = 67 cells, 9 slides) in the DPC-exposed cells versus 471 ± 30 nM (n = 69 cells, 9 slides) in vehicle-exposed cells (P < 0.001). Resting Ca2+ levels were not significantly different between the two groups (Figure 4B).


Figure 4
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  		Figure 4. Effect of CFTR blockers on the peak Ca2+ release induced by histamine in non-CF ASM cells. Exposure to glibenclamide (100 µM) and to N-phenylanthranilic acid (DPC; 100 µM) significantly reduced Ca2+ release evoked by 10–4 M histamine. (A) Open bars, vehicle; hatched bars, glibenclamide. (B) Open bars, vehicle; hatched bars, DPC. Data are shown as means + SEM.

 
Effects of Inhibiting CFTR Protein Expression Using Antisense–ODNs
To confirm that glibenclamide inhibition of the Ca2+ release induced by 10–4 M histamine was due to reduced CFTR activity, and not to other pharmacological effects of glibenclamide, histamine-induced Ca2+ release was measured in non-CF ASM cells that had been exposed for 72 hours to either vehicle, FuGENE 6, or to FuGENE 6 plus sense-ODNs or antisense-ODNs. Peak [Ca2+]i release was significantly lower in cells lacking CFTR compared with the other three groups of cells (three independent experiments for each group) (i.e., 151 ± 12 nM versus 243 ± 12 nM in vehicle-exposed cells [n = 33 and 37 cells, respectively; P < 0.001]; 249 ± 15 nM in FuGENE 6–treated cells [n = 37 cells; P < 0.001] and 204 ± 14 nM in cells exposed to sense-ODNs [n = 37 cells; P = 0.005]) (Figure 5).


Figure 5
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  		Figure 5. Inhibition of CFTR expression decreases Ca2+ release by histamine. Peak Ca2+ release was significantly lower in cells lacking CFTR compared with the other three groups of cells: 151 ± 12 nM versus 243 ± 12 nM in vehicle-exposed cells (P < 0.001); 249 ± 15 nM in FuGENE 6–treated cells (P < 0.001); and 204 ± 14 nM in cells exposed to sense-oligodeoxynucleotides (ODNs) (P = 0.005). H, peak Ca2+ release after 10–4 M histamine stimulation; R, resting Ca2+ levels. Open bars, vehicle; hatched bars, fugene; cross-hatched bars, sense; solid bars, antisense. Data are shown as means + SEM.

 
In Figure 6, CFTR localization by indirect immunofluorescence shows that CFTR was clearly down-regulated in the cells treated with antisense-ODNs (Figure 6B) compared with the cells treated with sense-ODNs (Figure 6A). Figure 6C is a bright-field microscopy picture of the image shown in Figure 6B. The appropriate negative controls, in which exposure to the primary antibody were omitted, were performed and showed no nonspecific staining.


Figure 6
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  		Figure 6. Effects of antisense-ODNs on CFTR expression in ASM cells. CFTR was clearly down-regulated in the cells treated with antisense-ODNs (B) compared with the cells treated with sense-ODNs (A). (C) Bright field microscopy picture of the image shown in (B). Scale bars = 50 µm.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 Statistical Analysis
 RESULTS
 DISCUSSION
References
 
Defects in CFTR have profound effects on epithelial cell function, and abnormal airway function in CF-affected persons is usually interpreted as resulting from these effects. In this study, we demonstrate that CFTR channels are expressed in human ASM cells, and propose that defective function in this location may also have important pathophysiological consequences. Wide-field illumination was used to maximize the signal from indirect immunofluorescence staining, which, although precluding precise cellular localization, nevertheless suggests a diffuse distribution of the CFTR channels as in other nonepithelial cells, such as lymphocytes, cardiomyocytes, endothelial cells, and rodent vascular and tracheal smooth muscle cells (14–16, 18). There are few studies of CFTR in tissues other than epithelium. Recently, activation of the CFTR channels by specific agonists in rat vascular and tracheal smooth muscle strips has been shown to induce relaxation of precontracted tissue, leading to the speculation that the relaxation is due to an inward flow of anions triggered by the opening of the CFTR channel. In turn, this inward flow promotes hyperpolarization of the cell, and counteracts the depolarization induced by the contractile agonist and the inward flow of Ca2+ ions (16). Such a mechanism could have important consequences for airway function if alterations in Ca2+ handling were indeed caused by defective CFTR and would be expected to result in enhanced smooth muscle contraction. A difference in Ca2+ regulation in response to histamine has been observed in tracheal gland cells and in nasal epithelial cells of patients with CF, but the reason for this abnormality was not explored (28). Considering that Ca2+ is an important regulator of smooth muscle constriction, and that an abnormal response to contractile agonist could contribute to the asthma diathesis observed in patients with CF, we measured the response of CF and non-CF ASM cells to the contractile agonist, histamine. CF ASM cells have a slightly lower resting [Ca2+]i, and the peak Ca2+ release in response to histamine is decreased. This decrease in peak Ca2+ response is not specific for histamine, as we also observed it in CF cells exposed to IL-8 (29) and UTP (unpublished data).

A potential role for Cl– channels, although not specifically CFTR, in the regulation of [Ca2+]i in ASM cells has been recently proposed by Janssen (30), who suggests that, after agonist exposure, Ca2+ is released from the sarcoplasmic reticulum, thus increasing the negative charge on the inner side of the membrane of the sarcoplasmic reticulum. This change in electrochemical gradient in turn hinders further release of Ca2+. However, the leak of Cl– from the sarcoplasmic reticulum into the cytoplasm diminishes the electronegativity, thus allowing more Ca2+ to be released. Accumulation of Cl– ions in the cytosol hinders both processes until the opening of Cl– channels on the plasmalemma allows the Cl– ions to diffuse out of the cell and permits the ongoing diffusion of Cl– from the sarcoplasmic reticulum, thereby enhancing Ca2+ release. This hypothesis is consistent with the present data, showing a decreased release of Ca2+ in response to agonist in CF smooth muscle cells. To determine if such a mechanism could operate in ASM cells, we first determined whether the difference in Ca2+ release in response to histamine was dependent on membrane channels by measuring the histamine-induced Ca2+ release in Ca2+-free extracellular medium. The results show that the difference between CF and non-CF ASM cells was abolished, thus indicating that it is attributable to ion exchange between the cells and the extracellular medium. The possible role of the CFTR channel was then assessed by using the standard CFTR pharmacological blocker, glibenclamide, at a concentration shown to effectively block Cl– channels (30). Exposure to glibenclamide slightly increased the resting [Ca2+]i, an increase possibly attributable to the fact that glibenclamide is also a blocker of KATP channels. Indeed, blocking the K channels could result in cell depolarization and the subsequent entry of extracellular Ca2+. Exposure to glibenclamide and to DPC, another Cl– channel blocker, decreased the peak release of Ca2+ in response to histamine, which strongly suggests that the CFTR channels are involved in the regulation of Ca2+ release from the sarcoplasmic reticulum through changes in electrochemical gradient.

Although the precise location of intracellular CFTR channels was not determined in our cultured ASM cells, it has been reported that functional CFTR channels are present in the endoplasmic reticulum (31), and Hirota and colleagues (32) recently showed that Cl– channels are present on tracheal smooth muscle sarcoplasmic reticulum. Also, Vandebrouck and colleagues (18) have demonstrated that the CFTR channel is functional in rat tracheal smooth muscle, although the diffuse pattern of CFTR immunostaining in rat tracheal smooth muscle did not indicate CFTR localization to the plasma membrane. To confirm that these effects of the pharmacological blockers on Ca2+ release are directly attributable to the inhibition of the CFTR channels, and not to other potential pharmacological effects of the drugs, we selectively inhibited CFTR protein expression by transfecting the cells with antisense oligonucleotides. The results show that Ca2+ release was lower in the cells transfected with antisense ODN compared with those treated with control sense-ODN, or with the transfection agent FuGENE 6, thus confirming that the CFTR channel is implicated in intracellular Ca2+ regulation. Considering that Ca2+ is one of the major determinants of smooth muscle contraction, these data suggest that the modulation of contraction is affected in CF smooth muscle cells. The role of various ion channels, including Cl– channels, has been investigated in various types of smooth muscle cells, and it is now widely acknowledged that the membrane potential of vascular smooth muscle cells is determined by the flow of ions through the various channels present on the plasma membrane. In ASM cells, on the other hand, the role of ion channels in modulating contraction and relaxation is not as clear, as both contraction and relaxation can be elicited in the presence of various ion channel blockers. Fortner and colleagues (33) report that the epithelium-dependent relaxation of precontracted murine trachea by substance P or ATP is inhibited by the Cl– channel inhibitor, DPC, suggesting that the CFTR channel may be involved; however, the relative importance of the epithelial versus smooth muscle CFTR channels in mediating the relaxation was not determined. Also, ATP- and SP-induced tracheal relaxation are enhanced when Cl– secretion by epithelial cells is increased, confirming a role for Cl– ions in the relaxation. Very recently, Hirota and colleagues (32) have shown that Cl– channel blockers inhibit peak ASM contraction. They attribute this decrease in contraction to the presence of Cl– channels on the sarcoplasmic reticulum, which neutralize charge accumulation, and thus modulate Ca2+ release and uptake, as discussed above. These observations were corroborated by Alapati and colleagues (34), who observed that the contraction of bovine arteries was decreased in Cl–-free medium. These observations are consistent with our data showing a decrease in peak [Ca2+]i release in response to contractile agonists. They are, however, difficult to reconcile with reports that CFTR activation promotes smooth muscle relaxation of rat tracheal and vascular smooth muscle. However, the experimental conditions are different. In the first case, the inhibition of the Cl– channel inhibits the development of peak contraction, presumably through interference with Ca2+ release. In the second case, the authors show the effect of CFTR stimulation once contraction has been fully established. In this latter case, the influx of anions would promote hyperpolarization of the cell, and counteract the depolarization induced by the contractile agonist.

In humans, the importance of CFTR regulation of smooth muscle contractility is not known. However, many of the clinical manifestations of CF that are attributed to abnormalities in mucus secretion and clearance, such as bronchoconstriction, airway hyperresponsiveness, gastric dysmotility, and intestinal obstruction, could be due, at least in part, to smooth muscle dysfunction. Indeed, the fact that ASM cells of patients with CF are hypercontractile to IL-8 (35), coupled to the fact that CFTR activation induces relaxation, lends credence to this hypothesis.

In conclusion, our data show that the CFTR channel is expressed and functional in human ASM cells, and is involved in intracellular Ca2+ regulation in response to a contractile agonist. We also demonstrate that intracellular Ca2+ homeostasis is affected in CF ASM cells. The importance of these observations in relation to the various clinical problems described in patients with CF remains to be determined.


   Footnotes
 
This work was supported by the Canadian Cystic Fibrosis Foundation and a McGill University Health Center Research Institute fellowship (R.R.).

Originally Published in Press as DOI: 10.1165/rcmb.2006-0444OC on August 28, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form December 1, 2006

Accepted in final form June 27, 2008


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 Statistical Analysis
 RESULTS
 DISCUSSION
 References
 

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