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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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The pulmonary disease of cystic fibrosis (CF) is characterized by persistent airway obstruction, which has been attributed to chronic endobronchial infection and inflammation. The levels of exhaled nitric oxide (NO) are reduced in CF patients, which could contribute to bronchial obstruction through dysregulated constriction of airway smooth muscle. Because airway epithelium from CF mice has been shown to have reduced expression of inducible NO synthase, we examined airway responsiveness and relaxation in isolated tracheas of CF mice. Airway relaxation as measured by percent relaxation of precontracted tracheal segments to electrical field stimulation (EFS) and substance P, a nonadrenergic, noncholinergic substance, was significantly impaired in CF mice. The airway relaxation in response to prostaglandin E2 was similar in CF and non-CF animals. Treatment with the NO synthase inhibitor NG-nitro-L-arginine methylester reduced tracheal relaxation induced by EFS in wild-type animals but had virtually no effect in the CF mice. Conversely, exogenous NO and L-arginine, a NO substrate, reversed the relaxation defect in CF airway. We conclude that the relative absence of NO compromises airways relaxation in CF, and may contribute to the bronchial obstruction seen in the disease.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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In cystic fibrosis (CF), defective function of the CF transmembrane conductance regulator (CFTR) in airway epithelial cells and submucosal glands results in chronic involvement of the respiratory tract, manifested by progressive airway obstruction that begins early in life (1, 2). Yet the precise mechanism in which the absence of a functional cyclic adenosine monophosphate chloride channel actually leads to pulmonary disease remains uncertain.
Failure of chloride secretion through CFTR or associated ion channels results in the dehydration of endobronchial secretions. Desiccated secretions block the airways and prevent elimination of bacteria (2). The lungs of neonates with CF are structurally normal with the exception of plugging and distension of the submucosal gland ducts (3, 4). Cultures of respiratory secretions from infants often fail to yield a specific pathogen (5). In addition, bronchial hyperreactivity is a common problem in CF (6), occurring in as many as 40% of affected individuals, which further contributes to the airway obstruction (8).
Recently, several investigators have demonstrated that exhaled nitric oxide (NO) levels are significantly reduced in patients with CF, a surprising observation because NO production would be expected to be increased in such an inflammatory disease (10). Lower NO metabolite levels have been correlated with poorer lung function in these patients, suggesting that lower NO production could be a marker of more severe lung disease (13). It is also possible that there may be a causal relationship between decreased production of NO and impaired lung function in CF.
We hypothesized that lower NO production in CF mice may lead to impaired relaxation of precontracted airway smooth muscle. To test this hypothesis, we compared the airway relaxant response of CF mice homozygous for the null S489X mutation with that of their normal littermates. These mice have been shown to have a marked reduction of nitrate production and inducible NO synthase (iNOS) in their airways (14). If NO deficiency actually contributes to the airway obstruction that is characteristic of CF, CF mice should have dysregulated airway smooth-muscle tone compared with their normal counterparts. We chose to use electrical field stimulation (EFS), prostaglandin (PG) E2, and substance P (SP) to study the relaxant response of precontracted trachea because NO is responsible for EFS-induced airway relaxation in vitro (15), and NO is also involved in the mechanism responsible for SP- and PGE2- induced airway relaxation (16, 17). We hypothesized that in CF, airway relaxation induced by EFS or SP is impaired, and the lack of NO contributes to this impairment.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animals
Male and female mice homozygous for the S489X (CFTR
/)
mutation of the CFTR and their normal littermates (CFTR+/+)
were studied (18, 19). The genotypes of individual animals were
established by polymerase chain reaction amplification of genomic DNA isolated from the animals' tails as previously described (20). Pups were weaned and fed liquid elemental diet
(Peptamen; Clintech Nutrition Co., Deerfield, IL) ad libitum beginning at Day 10 of life to avoid the intestinal obstruction characteristic in mice with this mutation (20). Sterile water was available at all times for control and experimental animals. All mice
were maintained in microisolator units with inedible bedding,
which has been shown to reduce the frequency of intestinal obstruction (19). Most CF mice reach adulthood and have grossly
normal lungs. Animals were studied when they were 8 to 12 wk old.
Experimental Preparation
CFTR+/+ and CFTR/ mice were killed and their tracheas removed. Under the dissecting microscope each trachea was freed
of adventitia and fat tissue. A cylindrical airway segment of 3 mm
length was isolated from the mid-trachea of each animal and
placed in a modified Krebs Henseleit solution of the following
composition (in mM): NaCl, 115; NaHCO3, 25; KCl, 2.5; NaH2PO4,
1.38; MgSO4, 2.46; CaCl2, 1.91; and dextrose, 5.56 with a pH adjusted to 7.4. The solution was continuously aerated with 5%
CO2 balanced with O2. Tracheal cylinders were suspended between a sturdy glass rod and a force displacement transducer (FT
03; Grass Instruments, Quincy, MA) connected to an amplifier as
previously described (21). Generated forces were continuously
monitored and recorded on a rectilinear chart recorder. The cylinders were allowed to equilibrate in the organ bath (Radnoti
Glass, Monrovia, CA) for 40 to 45 min before any challenge. The
optimal length at which maximal isometric force developed was
obtained for each cylinder by 0.1-g increments of load until EFS
(5 V DC applied though platinum electrodes, 250 mA/cm2) applied for 10 s at 4-min intervals gave a reproducible maximal response.
Experimental Protocol
A cumulative concentration-response curve to bethanechol (3 × 108 to 103 M) was obtained for both groups. The concentration
of bethanecol that elicited 50 to 75% of maximal response (ED50-
75) was determined. The airway cylinders were then washed,
equilibrated, and precontracted with bethanecol (ED50-75: between 3 × 106 and 105 M). EFS was then applied to each precontracted cylinder at a range of 0.5, 1, 2, 4, 8, 16, 32, and 64 Hz at
a constant voltage (5 V) and DC current. The percent relaxation
from the precontracted state was calculated for each cylinder.
The percent relaxation in response to EFS was calculated from
the reduction in tension (in g) in relation to baseline tension in
the precontracted state, each tracheal segment serving as its own
control (before and after EFS exposure).
To determine whether SP-induced airway relaxation is impaired in CF animals, precontracted airway segments of CFTR+/+
and CFTR/ animals were exposed to SP at 2 × 1010 M, the
concentration that induced the same percent relaxation seen with
EFS at high frequencies in wild animals. The percent relaxation
of precontracted trachea induced by SP was then calculated. In
additional CF and non-CF animals, we sought to block the relaxant effect of EFS on precontracted airways. Tracheal segments
were exposed to the NO synthase (NOS) inhibitor NG-nitro-L-arginine methylester (L-NAME) at 104 M for 30 min before bethanechol, followed by EFS stimulation as described earlier.
L-NAME preferentially blocks constitutive NOS isoform and
neuronal NOS (nNOS). Cylinders exposed only to bethanechol
followed by EFS served as controls. To determine the relaxant
mechanism underlying possible differences in response of airways to endogenously released NO, tracheal segments from CF
and wild-type animals were incubated with a precursor of NO, L-arginine, at 104 M for 30 min before exposure to bethanechol
followed by EFS stimulation. Precontracted trachea from both
CFTR/ and control groups were also exposed to exogenous
NO (105 M) solution, at a concentration that induced a similar
percent relaxation of airways seen with EFS at high frequencies.
To determine the effect of relaxant PGs on CF airways, precontracted trachea from CF and non-CF animals were exposed to
PGE2 (2 × 1010 M) at a concentration that induced a similar
percent relaxation seen with SP (2 × 1010 M) and EFS at high frequencies.
Drugs
All dilutions of drugs were prepared on the day of the experiments. Stock solutions were made by dissolving SP (Sigma) in distilled water at 1 mM. Bethanechol, L-NAME, and L-arginine were dissolved in distilled water. PGE2 was dissolved in 0.1 M phosphate buffer solution. The NO solution was prepared by bubbling nitrogen gas into 10 cc of distilled water in a vacuum container for 30 min, followed by addition of NO gas for a subsequent 30 min. Using an electrochemical method we determined that the concentration of NO in the final solution saturated with NO approximated 1 mM (22).
Statistical Analysis
Two-way analysis of variance (ANOVA) with repeated measures
was used for statistical analysis of CF and non-CF tracheal segment responses to EFS. Two-way ANOVA tests with repeated
measures were also used for comparison of EFS-induced relaxation in the presence and absence of L-NAME or L-arginine in
CF and non-CF animals. Unpaired t tests were used to analyze
the response to exogenous NO, SP, or PGE2 in CF and non-CF
animals. All data are expressed as means (+/
) standard error of
the mean (SEM).
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Results |
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Materials and Methods
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Discussion
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Tracheal Contraction and Relaxation in CF versus Non-CF Mice
In initial experiments, the responsiveness of excised tracheal segments from CF (n = 12) and wild-type (n = 9)
mice was examined. Increasing concentrations of bethanechol
produced a similar dose-dependent increase in percentage
of maximal tracheal tension (%T max) in both groups
(Figure 1A). However, in contrast to non-CF animals (n = 9), precontracted trachea from CF mice (n = 12) had a diminished relaxant response to EFS (Figure 1B; P < 0.02, two-way ANOVA with repeated measures). In contrast to
control littermates, precontracted airway segments from
CF animals also failed to relax to exogenous SP at 2 × 1010 M, a concentration that produces a similar relaxation
to EFS at high frequencies (Figure 2A).
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To determine whether impaired relaxation to SP in the
CF group is secondary to PGE2-mediated mechanisms, we
exposed precontracted tracheal segments to PGE2 at 3 × 109 M, a concentration that induces a comparable percent
relaxation to SP in control animals. Tracheas from CF and
non-CF animals had similar responses (Figure 2B).
Effects of L-NAME, L-Arginine, and NO on Airway Relaxation in CF Mice
In this series of experiments we sought to determine the role of NO in the impairment of airway relaxant responses in CF animals. Preincubation with L-NAME, a NOS inhibitor, occurred before EFS exposure in constricted airway segments from CF (n = 6 with L-NAME; n = 12 without L-NAME) and wild-type animals (n = 6 with L-NAME; n = 9 without L-NAME). L-NAME caused a significant decline in the degree of relaxation induced by EFS in wild animals (Figure 3; P < 0.007, two-way ANOVA with repeated measures). In contrast, pretreatment with L-NAME did not affect relaxation of precontracted tracheal cylinders in CF mice (Figure 3; P = 0.15, two-way ANOVA with repeated measures).
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We also sought to determine whether deficiency of the precursor of NO, L-arginine, might contribute to decreased airway relaxation in CF animals. Preincubation with L-arginine corrected the relaxation defect seen in tracheas from CF animals. EFS-induced airway relaxation of precontracted trachea improved significantly in the presence of L-arginine in CF animals (Figure 4; n = 6 for the group incubated with L-arginine; n = 12 for the control group; P < 0.05, two-way ANOVA with repeated measures). Tracheal segments from wild-type animals did not relax further in response to EFS in the presence of L-arginine (Figure 4; n = 7 for the group treated with L-arginine; n = 9 for the control group).
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To determine whether the CF animals exhibited impaired relaxant responses to exogenously administered NO, we exposed precontracted airway segments to NO. Tracheas from CF and non-CF mice had the same degree of relaxation after treatment with NO (Figure 5).
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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The current study demonstrates that tracheas from CF mice have impaired relaxation in response to EFS. This effect appears to be related to the lack of NO produced by the CF respiratory epithelium, and is readily reversible by treatment with exogenous NO or L-arginine. This observation is consistent with previous findings of decreased exhaled NO in patients with CF (10) and a reduction in NOS expression in CF murine and human airway epithelial cells (14). Therefore, we now have evidence for a functional deficit that appears to be a consequence of NO deficiency in a CF model.
Our results have shown that increasing concentrations of bethanecol produced a similar dose-dependent increase in percentage of maximal tracheal tension in mature CF and non-CF animals. Previous data from our group have demonstrated that cholinergic agonists activate the NO- cyclic guanosine monophosphate (cGMP) pathway, opposing contractile responses in piglets, which is consistent with our current findings in mice (23).
We and others (16, 17) have previously shown that SP, a tachykinin neuropeptide released from sensory nerve endings of airways in response to noxious stimuli, induces relaxation of precontracted airways via a mechanism involving PGE2 and NO. In our current study, relaxation of precontracted tracheal segments of CF mice was impaired in response to SP but not to exogenous PGE2 or NO. These observations suggest that the endogenous production of NO and PGE2 may be impaired in CF mice. Exhaled NO has been shown to be reduced in patients with CF (10) and there is a correlation between the decrease in NO metabolites and the severity of lung disease (13). EFS induces airway relaxation via stimulation of nonadrenergic, noncholinergic inhibitory pathways, and endogenous release of NO in human trachea (15). We have previously shown that under in vitro conditions, exogenous NO induces airway relaxation of precontracted tracheal segments in rats (16). In the current study, the nonspecific NOS inhibitor L-NAME did not affect the degree of airway relaxation elicited by EFS in CF animals as it did in the airways of their wild-type littermates, which is consistent with previous observations of lack of NO in CF. When we incubated CF tracheal segments with L-arginine (a NO substrate) the percent relaxation of the precontracted airways improved significantly, suggesting that deficiency of NO production could be responsible for the lack of relaxation seen in CF.
Different NOS isoforms have been found to play a role in the regulation of pulmonary vascular tone (24, 25). Endothelial NOS, nNOS, and iNOS have also been described to be present in airway epithelium (26, 27). iNOS expression has been found to be deficient in the airways of CFTR gene knockout mice (14). NOS expression has also been found to be deficient in CF human trachea (14). In our current study the impaired airway relaxation seen in CF mice was improved by L-arginine, suggesting that the enzyme necessary to convert L-arginine to NO is present. Our observations are not inconsistent with previous findings showing that NOS expression is reduced in CF murine and human airway epithelial cells (14). EFS may induce NO release via a mechanism directly involving nNOS activation (15). NOS has been described in nerve fibers originating from intrinsic neurons in the airways. The recovery in the CF airways in the presence of L-arginine suggests that NOS isoforms other than iNOS may contribute to NO production in airways of CF mice. Future studies are warranted to map the expression of different NOS isoforms in CF airways.
Several investigators have previously shown that despite the lack of iNOS expression, CF airways appear to be
hypersensitive to exogenous NO (14). Tracheas from
CFTR/ mice had a several-fold increase in cGMP production in response to sodium nitroprusside (a NO donor)
over tracheas from non-CF animals (14). The ability of exogenous NO to induce relaxation of CF tracheal segments
in our study suggests that the signaling pathways after NO
release in airways are intact under the conditions applied
in our experiments, and that the lack of endogenous NO is
responsible for the impaired relaxation in CF.
Autonomic dysfunction and aberration have been described in CF (28). Patients with CF manifest an increased cholinergic and 
-adrenergic sensitivity and a decreased 
-adrenergic activity, which has been associated
with increased airway reactivity in many airway diseases
(29, 30). The underlying mechanism for airway obstruction
has been difficult to study in CF because at the time of
their diagnosis most CF patients have bacterial colonization of their airways with Pseudomonas aeruginosa and
Staphylococcus aureus. The infectious process elicits an inflammatory response with the release of cytokines and
mediators, which would affect airway reactivity (1, 2, 31).
Infection induces airway hyperreactivity in humans and
impairs airway relaxation in many species (32). In our
study we have demonstrated for the first time in noninfected CF animals that airway relaxation is impaired in
response to the adrenergic and nonadrenergic, noncholinergic inhibitory stimulation, and that airway hyperreactivity
could be secondary to impairment of airway relaxation.
The relative absence of NO in the CF airway has potential implications in the development and progression of pulmonary involvement in this disease. Decreased production of NO may lead to impaired antimicrobial activity in the CF airway and contribute to bacterial colonization of the lower respiratory tract (33). The lack of NO has also been implicated in chloride and sodium transport through the CF epithelium, and could possibly contribute to some of the reported electrophysiologic abnormalities (1, 2, 34). In the present manuscript we showed that lack of NO may have another, clinically significant role in the pathogenesis of CF lung disease. Airway relaxation is impaired, which is directly related to the absence of NO in the CF airways. Our observations suggest that the CFTR gene defect could be responsible for the impairment of airway relaxation and subsequently airway obstruction in CF. We have also shown that in our CF model, relaxation of airways to exogenous NO is intact and yet the relaxant response to EFS is impaired, suggesting that endogenous release of NO is diminished and correctable by L-arginine supplementation. Future studies might quantify the amount of endogenously released NO in CF versus wild-type animals.
We speculate that NO deficiency in CF is related to the
deficiency of its substrate L-arginine. This is in agreement
with recent findings showing that L-arginine increases NO
production in isolated lungs of chronically hypoxic newborn pigs (35). Whether L-arginine deficiency might be
due to intestinal malabsorption or lack of its bioavailability at the cellular level is unknown. Future in vivo studies
are needed to determine whether supplementation of
L-arginine to the CF diet improves airway relaxation. In
vitro studies are also warranted to measure NO production in tracheal cell cultures obtained from CFTR/ and
wild-type mice in the presence and absence of L-arginine.
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Footnotes |
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Address correspondence to: Musa A. Haxhiu, M.D., Ph.D., Rainbow Babies and Childrens Hospital, Div. of Neonatology, 11100 Euclid Ave., Cleveland, OH 44106-6010. E-mail: mah10{at}po.cwru.edu
(Received in original form August 7, 2000 and in revised form November 3, 2000).
* Current address: Dept. of Pediatrics, Washington University School of Medicine, St. Louis, MO.Abbreviations: analysis of variance, ANOVA; cystic fibrosis, CF; CF transmembrane conductance regulator, CFTR; mice homozygous for the S489X mutation of the CFTR, CFTR
/ mice; normal littermates of
CFTR/ mice, CFTR+/+ mice; electrical field stimulation, EFS; inducible
NOS, iNOS; NG-nitro-L-arginine methylester, L-NAME; neuronal NOS,
nNOS; nitric oxide, NO; NO synthase, NOS; prostaglandin, PG; standard
error of the mean, SEM; substance P, SP; percentage of maximal tracheal tension, %T max.
This work was presented at the 1999 American Thoracic Society meeting.
Acknowledgments: This work was supported by NIH HL 56470, 50527 and DK48996.
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References |
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Discussion
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 H. Grasemann, F. Kurtz, and F. Ratjen Inhaled L-Arginine Improves Exhaled Nitric Oxide and Pulmonary Function in Patients with Cystic Fibrosis Am. J. Respir. Crit. Care Med., July 15, 2006; 174(2): 208 - 212. [Abstract] [Full Text] [PDF]
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H. Grasemann, R. Schwiertz, S. Matthiesen, K. Racke, and F. Ratjen Increased Arginase Activity in Cystic Fibrosis Airways Am. J. Respir. Crit. Care Med., December 15, 2005; 172(12): 1523 - 1528. [Abstract] [Full Text] [PDF]
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H. Grasemann, C. Grasemann, F. Kurtz, G. Tietze-Schillings, U. Vester, and F. Ratjen Oral L-arginine supplementation in cystic fibrosis patients: a placebo-controlled study Eur. Respir. J., January 1, 2005; 25(1): 62 - 68. [Abstract] [Full Text] [PDF]
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 P. Kc, C. A. Mayer, and M. A. Haxhiu Chemical profile of vagal preganglionic motor cells innervating the airways in ferrets: the absence of noncholinergic neurons J Appl Physiol, October 1, 2004; 97(4): 1508 - 1517. [Abstract] [Full Text] [PDF]
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