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2 Meakins-Christie Laboratories and Montreal Chest Institute Research Center, McGill University, Montréal, Québec, Canada H2X 2P2; and 1 Department of Clinical and Experimental Medicine, University of Ferrara, 44100 Ferrara, Italy
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Although it is well known that hypoxemia induces pulmonary vasoconstriction and vascular remodeling, due to the proliferation of both vascular smooth muscle cells and fibroblasts, the effects of hypoxemia on airway smooth muscle cells are not well characterized. The present study was designed to assess the in vitro effects of hypoxia (1 or 3% O2) on rat airway smooth muscle cell growth and response to mitogens (PDGF and 5-HT). Cell growth was assessed by cell counting and cell cycle analysis. Compared with normoxia (21% O2), there was a 42.2% increase in the rate of proliferation of cells exposed to 3% O2 (72 h, P = 0.006), as well as an enhanced response to PDGF (13.9% increase; P = 0.023) and to 5-HT (17.2% increase; P = 0.039). Exposure to 1% O2 (72 h) decreased cell proliferation by 21.0% (P = 0.017) and reduced the increase in cell proliferation induced by PGDF and 5-HT by 16.2 and 15.7%, respectively (P = 0.019 and P = 0.011). A significant inhibition in hypoxia-induced cell proliferation was observed after the administration of bisindolylmaleimide GF-109203X (a specific PKC inhibitor) or downregulation of PKC with PMA. Pretreatment with GF-109203X decreased proliferation by 21.5% (P = 0.004) and PMA by 31.5% (P = 0.005). These results show that hypoxia induces airway smooth muscle cell proliferation, which is at least partially dependent on PKC activation. They suggest that hypoxia could contribute to airway remodeling in patients suffering from chronic, severe respiratory diseases.
hypoxemia; protein kinase C; cell cycle; in vitro; cell growth
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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PROLONGED HYPOXEMIA IS EXPERIENCED frequently by persons suffering from advanced respiratory diseases and by high-altitude dwellers. Vascular remodeling is a well-known consequence of this environment and leads to pulmonary hypertension. Whereas a short exposure to hypoxia induces reversible constriction of the pulmonary vasculature, prolonged exposure is associated with the cellular and histological changes of vascular remodeling, and the vessels become resistant to vasodilators (23). The main structural changes induced by prolonged hypoxia in the vessels of pulmonary circulation are hypertrophy and hyperplasia of smooth muscle cells (SMCs) in the media and of fibroblasts in the adventitia (23, 44). Airway remodeling, including airway smooth muscle (ASM) hyperplasia, is also a feature of various pulmonary diseases, such as asthma (43), cystic fibrosis (45), and chronic obstructive pulmonary disease (27). The factors causing ASM hyperplasia have not been characterized but may include proinflammatory mediators to which the airways are exposed, as well as growth factors and components of the extracellular matrix (25). In addition, it is possible that tissue PO2 values may also play a role in the growth of ASM as well as its response to the mitogenic effects of growth-promoting substances.
The effects of hypoxia on pulmonary vascular cells have been extensively investigated. An increased proliferative response has been observed for pulmonary artery SMCs and fibroblasts exposed in vitro to low PO2 values. A peak response for pulmonary vascular SMCs has been observed at 3% O2, whereas lung fibroblast proliferation is also significantly increased at 1% O2 (15, 16, 38, 42, 48). To our knowledge, the direct effect of low PO2 on proliferation of ASM cells in vitro has not been investigated. As to the effects of hyperoxia, contradictory results have been reported, with an increase in cell proliferation in vivo and a decrease in vitro (1, 22). In a very recent paper by Pandya et al. (35), the relationship between PO2 and human fetal tracheal SMC cell proliferation has been examined and shows that O2 can regulate SMC proliferation also at a level of relative hyperoxia, 70 Torr compared with the in utero estimated PO2 of 38 Torr. If ASM cells respond to a hypoxic microenvironment with a proliferative response, this could have significant consequences for airway hyperresponsiveness. Indeed, an increase in muscle mass is likely an important determinant of the magnitude of airway narrowing in response to stimulation by contractile agonists (27, 29). A few reports suggest that chronic hypoxia in vivo can induce structural changes in the airways. Concomitant hyperplasia of both bronchial and vascular smooth muscle has been reported, both in animals experimentally exposed to high altitude and in humans with primary pulmonary hypertension and no history of chronic respiratory disease (19, 26, 31). Based on these studies, it has been postulated that the ASM hyperplasia is due to the same mediators acting on pulmonary vasculature.
We postulated that the proliferation of ASM cells in culture may be affected by the ambient O2 and that, under hypoxic conditions, these cells proliferate at a higher rate. We assessed the in vitro proliferation of rat ASM cells after exposure to two levels of hypoxia, 1 and 3% O2, as well as the interactions of hypoxia with concomitant stimulation with the multimitogenic stimulus, FBS, as well as more specific mitogens, PDGF and 5-HT. We studied also the role of PKC as a mediator of the effects of hypoxia on ASM cell growth. PKC is activated by both Ca2+ and diacylglycerol and is required for the proliferation of many types of cells. An involvement of PKC, in particular the Ca2+-regulated isozymes of this kinase, has been demonstrated in hypoxic proliferation of both fibroblasts and pulmonary artery SMCs (14, 17).
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Cell Culture
Primary cultures of ASM cells were prepared as previously described (49). Briefly, 7- to 9-wk-old male Fisher rats (Harlan Sprague Dawley, Walkerville, MD) were injected intraperitoneally with a lethal dose of pentobarbital, and the tracheas were removed and cut longitudinally through the cartilage and placed in HBSS. Tissue digestion was obtained by incubating the tracheas for 45 min at 37°C in HBSS containing 0.2% collagenase type IV and 0.05% elastase type IV. The dissociated cells were then collected by centrifugation; resuspended in culture medium containing 1:1 DMEM-Ham's F-12 medium supplemented with 10% FBS, 0.244% NaHCO3, penicillin (100 U/ml), and streptomycin (100 µg/ml); and plated in 25-cm2 culture flasks. Confluent cells were detached with a 0.25% trypsin-0.02% EDTA solution and grown on 24-well plates for cell counting. Cells from the first to fifth passage were used. They were identified as SMCs by positive immunohistochemical staining for smooth muscle-specific
-actin and
the absence of cytokeratin (49).
Effect of hypoxia on ASM cell growth rate. Confluent cells (1st to 5th passage) were detached by trypsinization, counted, and plated in 24-well plates at a density of 1 × 104 in DMEM-10% FBS. They were immediately exposed to either normoxia (21% O2, 5% CO2, balance N2) or hypoxia. For hypoxic stress, the cells were placed in a humidified Plexiglas chamber maintained at 37°C and continuously gassed with a gas mixture containing either 1 or 3% O2, 5% CO2, and balance N2. At the end of the experimental period (2, 3, or 4 days), the cells were detached with 0.5 ml of HBSS containing 0.25% trypsin and 0.02% EDTA. Cell growth was assessed by cell counting with a standard hemacytometer (American Optical, Buffalo, NY), and cell viability was assessed with Trypan blue. To limit the error that can be associated with counting, we followed the recommendation to count all of the cells loaded in all of the 9-mm2 squares of each side of the hemacytometer (24).
Effects of hypoxia on growth responses to mitogens.
Cells were plated as previously described and allowed to grow for
48 h in DMEM-10% FBS. After 48 h, the growth was arrested by
switching the culture medium to DMEM-0.5% FBS. Forty-eight hours
later, the mitogenic agents PDGF BB (10 nM) or 5-HT (10
4
M) or the same volume of diluent (100 µl DMEM-0.5% FBS) was added to
the medium, and the cells were allowed to proliferate for 48 h in
either normoxia or hypoxia (1 or 3% O2). All measurements were made in triplicate.
Cell Cycle Analysis in Normoxia vs. Hypoxia
First- to fifth-passage cells were plated in tissue culture flasks (75 cm2) and allowed to grow for 2 days in DMEM-10% FBS. In preliminary experiments, we varied the density of inoculation to obtain a confluence of ~40-50% after 48 h. The best results were obtained with an initial number of cells of 3 × 106. After 48 h, cells were growth arrested with medium containing 0.5% FBS for 2 days. After synchronization of the cell cycle by growth arrest, the cells were exposed to either normoxia or hypoxia (3% O2). A PKC inhibitor (see below) was added to one-half of the hypoxia-exposed cells. Fourteen hours later, the cells were detached by trypsinization; DMEM-10% FBS was then added to inactivate the trypsin. The cells were counted, centrifuged for 10 min at 1,200 rpm, and resuspended in Dulbecco's PBS at a density of 1-1.5 × 106/ml. Cells were centrifuged again, and 1 ml of 70% ethanol (
20°C) was added while the cells were
gently vortexing. The cells were maintained in ethanol for 1 h at
4°C and then centrifuged at 3,000 rpm. The ethanol was then poured
off, and the cells were stained for 30 min with modified Krishan buffer
(0.1% sodium citrate, 0.02 mg/ml RNase, 0.3% Nonidet P-40, 0.05 mg/ml
propidium iodide) (18, 49). Cells were analyzed for DNA
content in a flow-activated cell sorter. For each sample,
104 events were accumulated in a histogram. The proportions
of cells in the different phases of the cell cycle were calculated from each histogram.
PKC Inhibition
Two strategies to inhibit PKC activity were used: 1) enzyme inhibition using the PKC-Ca2+-dependent isozyme inhibitor bisindolylmaleimide (GF-109203X) and 2) PKC downregulation.Enzyme inhibition.
Cells were seeded in 24-well plates, as previously described, allowed
to grow for 48 h, and then growth arrested with DMEM-0.5% FBS for
48 h. GF-109203X was then added in increasing concentrations (107, 106, 5 × 106, and
105 M) to the culture medium, and the cells were exposed
to either 21 or 3% O2 for 48 h (46). At
the end of the experimental period, proliferation was assessed by cell counting.
PKC downregulation.
In preliminary experiments, the effects on cell proliferation of
pretreatment with different concentrations of PMA (5 × 107, 8 × 107, and 106
M) were tested, and no significant difference was found (data not
shown). Therefore, a concentration of 8 × 107 M was
used. Cells were plated and allowed to grow in normoxia in DMEM-10%
FBS and then growth arrested, as previously described. PMA was added 24 and 48 h after the beginning of the growth arrest period (culture
medium switched to DMEM-0.5% FBS), and cells were incubated in either
21 or 3% O2. Cells were counted 48 h later to assess
the extent of proliferation.
Chemicals
Elastase, collagenase, 5-HT, and PMA were purchased from Sigma Chemical (St. Louis, MO). GF-109203X was obtained from LC Services (Waltham, MA), and PDGF BB from ICN (Mississauga, ON). DMEM, FBS, penicillin, and streptomycin were from GIBCO Canada (Mississauga, ON); Ham's F-12 was from ICN; and EGTA from Calbiochem (San Diego, CA).Data Analysis
All data are expressed as means ± SE. Data were analyzed by paired t-test, with the rationale being that the cells are naturally clustered because cells from the same culture flask are assigned to each treatment. In statistical terms, it was a complete balanced block design.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Effects of Hypoxia on ASM Cell Growth
The effects of exposure to 1 and 3% O2 on ASM cell proliferation (concurrently incubated in 10% FBS) are shown in Fig. 1. The data show that, compared with normoxia, exposure to 3% O2 (corresponding to an actual PO2 of 38-40 Torr measured in preliminary experiments; see DISCUSSION) significantly enhanced cell proliferation, and that this effect was time dependent. After 48 h of exposure, cell counts were increased by 19.9 ± 4.5% (P = 0.028, n = 6). After an exposure of 72 h, the increase was 42.2 ± 6.7% (P = 0.006, n = 6) and 44.0 ± 9.5% after 96 h (P = 0.002, n = 6). Exposure to severe hypoxia (1% O2, corresponding to an actual PO2 of 10-15 Torr; see DISCUSSION) slowed down cell proliferation; cell counts were decreased by 27.5 ± 5.8% after 48 h of exposure (P = 0.007, n = 6). They were also significantly decreased by 21.0 ± 6.3% and 19.7 ± 5.1% after 72 and 96 h of exposure (P = 0.017 and 0.012; n = 6), respectively. Cell viability was 94.6 ± 0.6% in normoxia, 93.8 ± 0.5% in 3% O2, and 94.8 ± 0.5% in 1% O2.
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Effects of Hypoxia on the Growth Response of ASM Cells to Mitogenic Agonists
Figure 2A shows the effect of exposure to 3% O2 in baseline conditions (i.e., only vehicle added) and in cells treated with the mitogenic agonists PDGF BB (10 nM) or 5-HT (100 µM). In baseline conditions, exposure to 3% O2 produced a significant increase in cell proliferation compared with normoxia (5.73 ± 0.60 vs. 4.62 ± 0.40 × 104; P = 0.023; n = 10) and enhanced the effects of PDGF BB (10.86 ± 1.30 vs. 9.53 ± 1.07 × 104; P = 0.023; n = 10) and of 5-HT (100 µM) (7.26 ± 0.69 vs. 6.15 ± 0.60 × 104; P = 0.039; n = 10).
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Figure 2B shows the effects of exposure to 1% O2 in baseline conditions and in cells treated with PDGF BB and 5-HT (100 µM). In PDGF BB-exposed cells, exposure to 1% O2 significantly decreased cell proliferation (8.51 ± 1.00 vs. 10.13 ± 1.50 × 104; P = 0.019; n = 8). As well, the proliferation induced by exposure to 5-HT was also reduced (5.87 ± 0.73 vs. 6.96 ± 0.70 × 104; P = 0.011; n = 8).
Effect of PKC Inhibition
Figure 3A shows the effects of decreasing concentrations of the PKC inhibitor GF-109203X on cell proliferation in normoxic and hypoxic (3% O2) conditions. As not all concentrations of the inhibitor could be tested at the same time, resulting in several different experiments, the results are expressed as a percentage of control cell (incubated in vehicle only) counts in normoxia. After exposure to 10 µM GF-109203X, cell counts were significantly decreased in both normoxia (P = 0.002, n = 6) and hypoxia (P = 0.003, n = 6). At concentrations of 5 and 1 µM, GF-109203X abolished the increase in cell counts due to exposure to 3% O2 (P = 0.0002 and 0.004, respectively) but had no significant effect on normoxic cells (n = 6). The results obtained after PKC downregulation are shown in Fig. 3B. Pretreatment with 0.8 µM PMA abolished the enhanced proliferation induced by 3% O2 and did not affect cell proliferation in normoxia. The mean cell number in control wells was 4.36 ± 0.50 × 104 in normoxia and 6.04 ± 0.88 × 104 in hypoxia (P = 0.042, n = 6). After pretreatment with PMA, the cell counts were 4.00 ± 0.47 × 104 in normoxia and 4.11 ± 0.72 × 104 in hypoxia. Compared with the control wells, the decrease in cell counts produced by PMA in 3% O2-exposed cells was highly significant (P = 0.010).
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Cell Cycle Analysis in Normoxia vs. Hypoxia
The effects of exposure to 14 h of either hypoxia (3% O2) or normoxia on the cell cycle are shown in Fig. 4. The data show that, in normoxia, 81.9 ± 2.2% of the cells were in the G0-G1 phase of the cycle, 3.1 ± 1.0% in the G2-M phase, and 14.9 ± 7.0% in the S phase (n = 7). In hypoxia, the proportion of cells in the G0-G1 phase was significantly lower than in normoxia (70 ± 2.4%; P = 0.009). It was comparable in the G2-M phase (2.8 ± 1.4%) and increased in the S phase (29.2 ± 3.3%; P = 0.008). After pretreatment of hypoxic cells with GF-109203X, the differences between hypoxic and normoxic cells were abolished (n = 6).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The ambient PO2 can variably affect cell growth. A higher rate of proliferation during hypoxic exposure is a feature of some but not all cells; vascular SMCs and fibroblasts in culture grow better at low PO2 (3, 8, 21, 33), whereas other cells of other origins (synovial and human cervical carcinoma cells) show a depression of growth, becoming quiescent during hypoxic stress (2, 39, 40). The effect of hypoxia on ASM cells has not been investigated as thoroughly as on vascular smooth muscle. In addition, the role of hypoxia in airway hyperresponsiveness has not been clearly elucidated.
It has been reported that hypoxia acts as a mitogenic agent on several cell types, including mesangial cells, vascular SMCs, and fibroblasts (7, 33, 41). In ASM cells, most studies have examined the effects of hypoxia on airway responsiveness to contractile agonists. In vitro studies report either an increase (6, 13) or a decrease in airway responsiveness (9) after exposure to hypoxia. Similar results were obtained in vivo, with some authors showing an increase in airway responsiveness (13) and others not (10). The different periods of exposure (very short vs. prolonged) and the different levels of hypoxia could, at least in part, explain these contradictory results. Because circulating catecholamines and cortisol levels are increased in subjects at high altitude [this is true especially for people usually resident at low altitude when exposed to high altitude (46a)], a direct effect of hypoxia on ASM may be difficult to detect. Furthermore, there are no morphological studies about remodeling in airways from high-altitude residents. Thus the aim of this study was to determine the effect of hypoxia on cultured ASM cells, isolated from neurohumoral stimuli, to determine whether it enhances ASM cell proliferation and, consequently, could cause airway remodeling. The choice of O2 concentrations to which the cells were exposed, 1 and 3%, was based on those used to study vascular smooth muscle responses to hypoxia (11, 20). It should be noted that, although 3% O2 corresponds to a calculated PO2 of 21 Torr, ambient PO2 in the culture flasks ranged between 38 and 40 Torr after 48 h of incubation (data not shown). Similar discrepancies have been published for 1% O2 with calculated values of 7 Torr and measured values ranging from 10 to 15 Torr (19) and for 0% O2 with values ranging from 18 to 20 Torr after a few hours of incubation (12, 30). This appears to be caused by the slow equilibration between the original PO2 in the culture wells and the PO2 of the gas mixture employed (28). This range of PO2 values is likely to be comparable to values encountered in pathophysiological conditions.
The results that we obtained show that ASM cells exposed to severe hypoxia (1% O2) for 48 h are still able to grow but at a slower rate than cells grown in normoxia (21% O2). The observation is not limited to ASM cells, as Cooper and Beasley (11) report very similar results for the effects of severe hypoxia (1%) on vascular SMCs. This slower proliferation rate, present in cells exposed to severe hypoxia only, was maintained in the presence of mitogenic agents such as PDGF and 5-HT. This lower cell count observed under conditions of severe hypoxia does not appear to be due to apoptosis, because cell viability showed no difference between normoxic and hypoxic cells. Thus these results are similar to other published data showing that severe hypoxia induces a state of quiescence in many cells (2, 3, 5).
Under conditions of moderate hypoxia (3% O2), the cells proliferated more rapidly than cells exposed to 21% O2. These results are consistent with a recent publication by Pandya et al. (35), who showed that the proliferation rate of human fetal ASM cell was higher at 75-Torr PO2 than at 140-Torr PO2. This increased proliferation rate was observed under baseline conditions and in the presence of both PDGF and 5-HT. Previous studies have demonstrated that various types of cells proliferate in response to hypoxia, with a maximal response at 3% O2. They include some subpopulations of vascular SMCs, glomerular mesangial cells, and adventitial fibroblasts (38, 41, 44). The observation that the addition of mitogens did not significantly increase the growth difference between normoxic and hypoxic cells suggests that hypoxia has a direct effect on cell proliferation. These effects were analyzed further by performance of a cell cycle analysis. The data that we obtained show that the distribution of normoxic cells in the different phases of the cell cycle is similar to that reported in other studies by using low or serum-free medium (49). In cells exposed to hypoxia, the number of cells in the G0-G1 phase of the cycle was decreased, and the number of cells in the S phase of the cycle was increased, suggesting that hypoxic cells have a faster cell cycle than normoxic ones.
We aimed also to clarify the role that PKC might have in mediating the
response of the cells to hypoxia. More and more evidence has been
accumulated in recent years about the role of PKC isozymes in ASM cell
proliferation and differentiation (32, 34, 36, 37, 50).
PKC is an intracellular signal-regulated kinase involved in many cell
functions, frequently in the intracellular transduction of
proliferative signals. In ASM cells, many PKC isoenzymes, both calcium
dependent and independent, have been identified (47). In
1991, Dempsey first hypothesized and demonstrated a possible role of
PKC activation as a requisite step for pulmonary artery SMC
proliferation in hypoxic conditions (17). Further work by other researchers showed that hypoxia, in the absence of serum or
mitogens, is able to specifically activate PKC and mitogen-activated protein kinase (21, 41, 42, 44). In our study, two
approaches were used to examine the role of PKC in mediating
hypoxia-induced proliferation: the addition of a PKC inhibitor and the
downregulation of PKC with PMA. Cells were incubated under normoxic and
hypoxic condition (3% O2) in the presence or absence of
the PKC inhibitor GF-109203X, an inhibitor with relative specificity
for the calcium-dependent PKC isozymes. At concentrations 
1 µM,
this inhibitor is specific for PKC and does not affect SMC
proliferation induced by intracellular mechanisms other than the PKC
pathway. The results that we obtained show that, after 48-h exposure to
hypoxia, proliferation was blunted in GF-109203X-pretreated cells.
These data are confirmed by the results obtained with PMA that show
that the hypoxia-induced proliferation was abolished by PKC downregulation.
We did not test the effect of inhibiting PKC on the decrease in proliferation observed in ASM cells exposed to 1% O2 for the following reasons: if, as we have shown, the stimulation of PKC is involved in the enhanced growth rate observed at 3% O2, then an inhibition of PKC might be required to explain inhibition of growth at 1% O2. The use of an inhibitor would not be a useful strategy to address this latter question. Furthermore, under normoxic conditions, inhibition of PKC caused a reduction in cell growth only at the highest concentrations of the inhibitor. This suggests that constitutive activity of PKC is not critical for growth under these conditions. We reasoned that, at high concentrations of inhibitor, nonspecific effects were more likely. We thought it unlikely that reduction in growth in 1% hypoxia was, therefore, attributable to reduced PKC activity.
In conclusion, these results show that exposure to a moderate level of hypoxia acts as a mitogenic stimulus for ASM cells. The enhanced growth is mediated, at least in part, by specific, calcium-dependent PKC isozymes. These data suggest that the tissue hypoxemia encountered in pathophysiological conditions could contribute to airway remodeling by modulating ASM cell growth. These findings may be of particular relevance for inflammatory airway diseases in which concomitant stimulation of ASM by growth factors may be occurring.
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ACKNOWLEDGEMENTS |
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We thank Patrice Vaillancourt for expert technical help.
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FOOTNOTES |
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This study was supported by an operating grant from the Canadian Cystic Fibrosis Foundation.
Address for reprint requests and other correspondence: J. G. Martin, Meakins-Christie Laboratories, McGill University, 3626 St Urbain St., Montréal, PQ, Canada H2X 2P2 (E-mail: James.Martin{at}mcgill.ca).
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.
10.1152/japplphysiol.00363.2002
Received 24 April 2002; accepted in final form 5 December 2002.
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TOP
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METHODS
RESULTS
DISCUSSION
REFERENCES
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