Departments of 1 Pediatrics and 3 Physiology and Biophysics and 2 Center for Human Genetics, Department of Genetics, Case Western Reserve University, Cleveland, Ohio 44106
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ABSTRACT |
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Transepithelial ion transport is regulated by a
variety of cellular factors. In light of recent evidence that nitric
oxide (NO) production is decreased in cystic fibrosis airways, we
examined the role of NO in regulating sodium and chloride transport in murine nasal epithelium. Acute intervention with the inducible NO
synthase (iNOS)-selective inhibitor
S-methylisothiourea resulted in an
increase of amiloride-sensitive sodium absorption observed as a
hyperpolarization of nasal transepithelial potential difference. Inhibition of iNOS expression with dexamethasone also hyperpolarized transepithelial potential difference, but only a portion of this increase proved to be amiloride sensitive. Chloride secretion was
significantly inhibited in C57BL/6J mice by the addition of both
S-methylisothiourea and dexamethasone.
Mice lacking iNOS expression [NOS2(/
)] also
had a decreased chloride-secretory response compared with control mice.
These data suggest that constitutive NO production likely plays some
role in the downregulation of sodium absorption and leads to an
increase in transepithelial chloride secretion.
cystic fibrosis; cystic fibrosis transmembrane conductance regulator; inducible nitric oxide synthase
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INTRODUCTION |
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NITRIC OXIDE (NO) is a powerful signaling agent shown to influence several cellular mechanisms. In this study, we explored the role of NO in regulating the balance between sodium absorption and chloride secretion across airway epithelial cells. The regulation of ion transport by NO in the respiratory system gains significance in light of recent evidence that NO is constitutively expressed by airway epithelial cells (11) and that NO production is decreased in exhaled air from the upper airways of cystic fibrosis (CF) patients (2, 6, 9, 25). Additionally, we have evidence that NO production is reduced in the airways of CF mice through a loss of expression of the inducible form of NO synthase (iNOS) in airway epithelium (19, 26). These mice carrying various CF-related mutations exhibit reduced NO production in response to lipopolysaccharide (LPS), reduced bactericidal activity in the lungs, and altered cGMP production in response to NO-donating compounds. Because NO has a wide range of effects in different systems, it is possible that the lack of NO production in CF airways contributes to CF-related dysfunction in epithelial ion transport. Aberrant ion transport properties that are characteristic of CF include a lack of cAMP-stimulated chloride secretion, a lack of basal gradient-driven chloride secretion as measured by the nasal potential difference assay, and an increase in the rate of amiloride-sensitive sodium absorption (21-23). Although the loss of cAMP-stimulated chloride transport has been established to be directly related to the loss of CF transmembrane conductance regulator (CFTR) activity (1), the mechanisms underlying the loss of basal chloride transport and increased sodium absorption are not clearly delineated. We set out to explore how a loss of NO production by airway epithelial cells would influence these two areas of ion transport regulation and to determine whether decreased NO contributes to CF-related ion transport abnormalities.
Chloride transport relevant to disease, whether basal or activated, in airway epithelia likely involves the activity of other chloride channels in addition to CFTR (5). Kamosinska et al. (16) have shown that endogenous NO stimulates a CFTR-independent chloride conductance in A549 human lung epithelial cells through a cGMP-dependent mechanism via the activation of soluble guanylate cyclase activity by NO. These data suggest that a loss of NO production in CF airway epithelia may result in the loss of a secondary chloride secretory response, further compounding problems caused by the lack of CFTR-mediated chloride secretion.
In addition to the stimulation of CFTR-independent chloride secretory pathways, NO has been shown to downregulate sodium reabsorption. Although the exact mechanisms of inhibition are not well understood, both Na+-K+-ATPase from the porcine cerebral cortex (31) and the Na+/H+ exchanger in rabbit proximal tubules (29) have been shown to be inhibited by NO-dependent mechanisms. However, NO has been reported to stimulate Na+-K+-ATPase activity in human corpus cavernosum smooth muscle, indicating that NO-dependent regulatory mechanisms are not well understood and that there may be tissue-specific differences in these regulatory pathways (13). NO is known to stimulate the production of cGMP through the activation of soluble guanylate cyclases, and cGMP is a well-studied signaling agent that has been shown to reduce the rate of amiloride-sensitive sodium absorption in several systems (30). As an example of this regulatory system, atrial natriuretic peptide stimulates cGMP formation via activation of guanylate cyclase A and causes a decrease in amiloride-sensitive sodium absorption in renal inner medullary collecting duct cells (24). We have demonstrated identical regulation of sodium transport using C-type natriuretic peptide (CNP) in nasal and tracheal epithelium of mice (17, 18), although a similar report looking at nasal epithelial cells obtained from scrapings from human subjects found no effect of CNP on either chloride or sodium transport (8). In this study, we explored whether NO may be a vital component in the normal regulation of basal sodium and chloride transport across epithelial cells and the possibility that a loss of this regulatory system in CF may further complicate ion transport abnormalities associated with a loss of CFTR activity.
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MATERIALS AND METHODS |
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Measurement of mouse nasal transepithelial potential difference values. Mouse nasal transepithelial potential difference (TEPD) was measured by the protocols of Grubb et al. (10) and Kelley et al. (20). Briefly, mice were anesthetized with 10 µl/g body wt of (in mg/ml) 0.4 acepromazine, 11 ketamine, and 2 xylazine in PBS. PE-10 tubing drawn out to approximately one-half of its original diameter was inserted 2-3 mm into the nostril of the mouse. Solutions were perfused at room temperature with a Razel A-99 syringe pump (Razel Scientific Instruments, Stamford, CT) at a rate of ~7 µl/min. A series of valves was used to change solutions, with a delay time of ~45 s between solution change and solution contact with the nasal epithelium. Ringer solutions consisted of chloride-replete HEPES-buffered Ringer containing (in mM) 10 HEPES, pH 7.4, 138 NaCl, 5 KCl, 2.5 Na2HPO4, 1.8 CaCl2, and 1.0 MgSO4, and chloride-free HEPES-buffered Ringer containing (in mM) 10 HEPES, pH 7.4, 138 sodium gluconate, 5 potassium gluconate, 2.5 Na2HPO4, 3.6 hemicalcium gluconate, and 1.0 MgSO4. All chemicals are from Sigma (St. Louis, MO).
iNOS-specific immunostaining. Nasal epithelia were excised from mice intraperitoneally injected with either dexamethasone (45 mg/kg body wt) or PBS, paraffin blocked, and sectioned. Sections were deparaffinized, solubilized in ice-cold methanol for 5 min, and placed in 2% goat immunoglobulin in PBS for 2 h. Antibody against mouse and human iNOS was obtained from Calbiochem (La Jolla, CA) and incubated with the samples at 4°C overnight at a dilution of 1:300 in PBS. Samples were washed 4 times in PBS for 10 min/wash. Goat anti-rabbit IgG conjugated to alkaline phosphatase was diluted 1:200 in PBS and incubated with samples for 2 h at 37°C. Samples were washed as before and stained for 20 min in Vector Red from Novacastra Laboratories (Newcastle, UK) according to the manufacturer's instructions, and slides were counterstained with hematoxylin. Quantitation of staining was performed with ImagePro imaging software.
Direct NO measurement from 9/HTEo
cells. 9/HTEo
cells were grown at 37°C in
95% O2-5%
CO2 to confluency in Dulbecco's
modified Eagle's medium with 10% fetal bovine serum, 2.5 mM
L-glutamine, and 150 µg/ml
hygromycin on Vitrogen-coated 30-mm tissue culture plates. For studies
of NO production, cells were treated with either dexamethasone (1 mM)
or vehicle (PBS) for 16 h before assay, and LPS (5 µg/ml) from
Pseudomonas aeruginosa (Sigma) was
added to the medium of all samples 5-7 h before assay. NO assays
were performed with an Iso-NO nitric oxide meter utilizing the 2 mM Iso-NOP NO sensor (World Precision Instruments, Sarasota, FL). Measurements were taken at 37°C by placing both the NO sensor and
samples in an incubator. Data were recorded on a MacLab/4e data-acquisition system from Advanced Instruments. The Iso-NO meter was
calibrated according to manufacturer's instructions using the chemical
method of NO production.
Mice. Pure C57BL/6J mice and
mice lacking iNOS expression [NOS2(/
)] were
obtained from Jackson Laboratories. Mice homozygous for the
F508
CFTR mutation and C57BL/6 and 129/Sv mice used to breed
second-generation mice of mixed backgrounds (B6129F2) were obtained
from Dr. Kirk Thomas (University of Utah School of Medicine). CF mice
were fed a liquid diet as described by Eckman et al. (7). Mice were
cared for in accordance with Case Western Reserve University Institutional Animal Care and Use Committee guidelines.
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RESULTS |
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Effect of iNOS inhibition on basal sodium and chloride
transport. To assess the effects of iNOS inhibition on
sodium transport, the iNOS-selective inhibitor
S-methylisothiourea (SMT; 100 µM) was perfused into the nasal passage of mice in the absence of amiloride, and the effect on TEPD was measured (3, 14, 27). The initial
decline in TEPD magnitude in the absence of amiloride seen in Fig.
1 is consistent with a previous report
describing murine nasal TEPD assays (10). A plateau value was reached
before the addition of SMT. The addition of SMT results in an ~5-mV
hyperpolarization of lumen negative TEPD (Fig. 1). The addition of
amiloride (100 µM) abolishes the effect of SMT and results in an
immediate depolarization of TEPD. Similarly, pretreatment with either
amiloride or the cGMP analog 8-bromo-cGMP (8-BrcGMP; 100 µM) prevents
the effects of SMT on nasal TEPD, showing that changes in TEPD mediated
by SMT are amiloride sensitive and a result of cGMP-dependent pathways. These data are consistent with the notion that constitutively produced
NO mediates a downregulatory effect on amiloride-sensitive sodium
absorption through the stimulation of guanylate cyclase activity and
cGMP production.
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To assess whether iNOS-generated NO has a role in regulating basal
chloride transport as well as sodium transport, we assayed C57BL/6J mice by the nasal TEPD method. C57BL/6J mice
have consistently large changes in TEPD (>5-mV hyperpolarization of
lumen negative TEPD) in response to perfusion into the nasal passage of
chloride-free Ringer solution that has gluconate-containing salts
substituted for chloride-containing salts. This perfusion in
association with the nasal TEPD assay increases the electrochemical
driving force for secretion of chloride into the lumen from nasal
epithelial cells and can be used as an approximation of basal
activation of chloride transport pathways. These same mice were
reassayed after a minimum 48-h recovery period with SMT (100 µM)
added to the perfusion solutions. In the presence of SMT,
hyperpolarization of lumen negative TEPD in response to chloride-free
Ringer solution is reduced from 9.0 ± 1.5 to 2.8 ± 0.3 mV
(n = 4 mice;
P = 0.009; Fig.
2A) 2.75 min after the switch to chloride-free Ringer solution, representing a
69% reduction in response. In B6129F2 mice that consistently lack a
significant response to chloride-free Ringer solution (<5-mV
hyperpolarization of lumen negative TEPD), the NOS substrate
L-arginine was added to the
perfusion solution. When mice were treated with
L-arginine (100 µM), changes
in nasal TEPD increased from 1.3 ± 0.5 to 6.9 ± 1.1 mV
(n = 10 mice;
P = 0.0004; Fig.
2B). These data demonstrate that
basal NO production is a vital part of the regulatory mechanisms
controlling chloride transport, although whether this NO-mediated
chloride transport is CFTR dependent still needs to be examined.
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Effect of dexamethasone on transepithelial ion
transport. It has been shown in several systems that
glucocorticoid treatment downregulates iNOS expression and NO
production (4, 32, 33). It was our goal to determine whether
dexamethasone would mimic the effects of the iNOS-selective inhibitor
SMT in modulating both chloride and sodium transport. Sodium transport
was indirectly monitored by measuring baseline TEPD values in mice
before, during, and after intraperitoneal injection of dexamethasone
(45 mg/kg body wt). Baseline values before treatment were 6.2 ± 0.8 mV (n = 5 mice) and
increased to
11.5 ± 1.0 mV
(n = 5 mice) with dexamethasone (Fig.
3A).
Baseline TEPD was depolarized by amiloride to a plateau value of
4.9 ± 1.2 mV before treatment but only to
8.2 ± 1.3 mV (n = 9 mice;
P = 0.05) after treatment with
dexamethasone (Fig. 3B). Although
the percentage of baseline TEPD sensitive to amiloride increased from
21 to 29% in the presence of dexamethasone, these data indicate that
the majority of the dexamethasone-mediated increase in baseline TEPD is
due to an amiloride-insensitive pathway.
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To examine the effects of dexamethasone on chloride transport, the
response to chloride-free Ringer solution was tested before and after
intraperitoneal injection of dexamethasone. Mice before dexamethasone
treatment had a 9.4 ± 0.7-mV hyperpolarization of lumen negative
potential in response to chloride-free Ringer solution. After
dexamethasone treatment, the response to chloride-free Ringer solution
was reduced to a 2.8 ± 1.7-mV hyperpolarization
(n = 9 mice;
P = 0.007; Fig.
4). If NO production is indeed responsible for the stimulation of basal chloride secretion, it is likely that cGMP
is the messenger that stimulates this transport. To test the role of
cGMP in the stimulation of basal chloride transport, we examined the
effects of the general guanylate cyclase inhibitor methylene blue on
the response to chloride-free Ringer solution. Before treatment with
methylene blue (100 µM), mice demonstrated a 13.6 ± 1.8-mV
hyperpolarization of lumen negative TEPD in response to chloride-free
Ringer solution. In the presence of methylene blue, these same mice
exhibited only a 2.6 ± 0.7-mV hyperpolarization of TEPD
(n = 5; Fig.
5).
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Effect of dexamethasone on iNOS expression and NO
production. It has been previously reported that
dexamethasone prevents the expression of iNOS (4, 32, 33). To determine
whether dexamethasone treatment directly effects iNOS expression in
murine airway epithelia, we performed iNOS-specific immunostaining.
Nasal sections from mice treated with either dexamethasone (45 mg/kg body wt) or vehicle were obtained and stained for immunoreactive iNOS.
Mice treated with vehicle showed strong staining for iNOS in airway
epithelial cells, whereas dexamethasone-treated mice demonstrated
limited iNOS expression (Fig. 6). These
data are consistent with previously reported results and with the
effects of dexamethasone on transepithelial ion transport compared with the effects of the iNOS-selective inhibitor SMT.
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The effects of dexamethasone on the production of NO were also tested
with the human tracheal epithelial cell line 9/HTEo. 9/HTEo
cells stimulated with LPS from P. aeruginosa produced NO levels measured to be 967.7 ± 101.6 nM (n = 4 mice), whereas cells stimulated with LPS in the presence of dexamethasone (1 mM)
produced only 100.0 ± 28.9 nM (n = 4 mice, P = 0.003; Fig. 7). For comparison, 9/HTEo
cells not
stimulated with LPS had an NO level of 132.3 ± 11.7 nM
(n = 3 mice; data not shown). These data demonstrate that treatment with dexamethasone reduces
LPS-stimulated NO production to background levels.
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Ion transport characteristics of NOS2(/
)
mice. Through inhibition of enzymatic activity with the
iNOS-selective inhibitor SMT or through inhibition of protein
expression with dexamethasone, we have shown that influencing iNOS
activity or production has regulatory effects on transepithelial ion
transport. Therefore, mice lacking the expression of iNOS
should exhibit these same changes in ion transport regulation. We
compared the response to chloride-free Ringer solution using the nasal
TEPD assay in C57BL/6J mice and NOS2(
/
) mice that
have been backcrossed at least seven generations to the C57BL/6J
background. Wild-type C57BL/6J mice
(n = 8) possessed a large 12.1 ± 1.7-mV hyperpolarization of lumen negative potential in response to
perfusion with chloride-free Ringer solution. The NOS2(
/
)
mice, however, responded to this solution change with only a 1.8 ± 1.1-mV hyperpolarization (n = 8;
P = 0.0004; Fig.
8A).
These data are consistent with our data showing that inhibition of iNOS
activity with either SMT or dexamethasone reduces the chloride-free
response. As a comparison with other low-responder mice as shown in
Fig. 3, the NOS2(
/
) mice were reassayed with
L-arginine (100 µM) in the
perfusion. Unlike the B6129F2 mice that possessed a low chloride-free
response but responded to exogenously added
L-arginine, the
NOS2(
/
) mice still lacked a response to this agonist.
These data suggest that the response induced by
L-arginine in the B6129F2 mice
is indeed due to the stimulation of NO production through iNOS.
Although there is a striking decrease in the chloride-free response in the NOS2(
/
) mice compared with the wild-type
C57BL/6J mice, both the NOS2(
/
) and the wild-type
C57BL/6J mice have significant responses to forskolin,
suggesting that there is normal CFTR activity (data not shown).
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Baseline TEPD was not significantly different between the two groups of
mice. NOS2(/
) mice had a baseline potential value of
11.4 ± 1.1 mV (n = 8)
compared with
11.1 ± 0.9 mV for C57BL/6J mice
(n = 8; Fig.
8B). However, the baselines for the
C57BL/6J strain appear to be already elevated relative to
baselines of B6129F2 mice with mixed backgrounds (C57BL/6 and
129/Sv from the University of Utah). Mice
(n = 8) with a mixed background
averaged only a
7.8 ± 0.7-mV baseline. Any increase in
baseline TEPD that would possibly occur due to a lack of iNOS
expression may be masked by the elevated baseline of the background
strain. Baselines measured in the presence of amiloride, however, did
show an increased amiloride-insensitive TEPD in the
NOS2(
/
) mice. C57BL/6J mice have a baseline of
5.0 ± 0.4 mV (n = 9) in the
presence of amiloride compared with
8.6 ± 1.4 mV
(n = 9;
P = 0.02) without
amiloride (data not shown). This magnitude of increased
amiloride-insensitive TEPD is identical to that induced with
dexamethasone, suggesting that a lack of iNOS expression somehow
influences amiloride-insensitive TEPD by a currently unknown mechanism.
The influence of strain background on basal ion transport parameters is
an area that needs further examination.
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DISCUSSION |
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It has been postulated that the response to chloride-free Ringer solution represents basal chloride transport and that this response is dependent on CFTR activity (23). However, the exact regulatory mechanisms that govern the basal chloride-secretory response are still unknown. In this study, we examined the role of endogenous NO production in regulating basal chloride transport in murine nasal epithelia as well as the effects NO has on the regulation of amiloride-sensitive sodium absorption.
NO has been shown to be a key regulatory agent in controlling the basal chloride current in A549 airway alveolar epithelial cells through a non-CFTR-mediated pathway. Kamosinska et al. (16) demonstrated that constitutive production of NO regulates tonic transepithelial chloride transport through the activation of soluble guanylate cyclases. Given recent results suggesting that NO production is diminished in CF airway epithelial cells, we wanted to examine whether a lack of NO production in vivo would effect basal chloride transport. We examined the effects of iNOS-selective inhibition on the chloride-free response in C57BL/6J mice. We previously reported that mice of mixed 129/Sv and C57BL/6 backgrounds had differential responses to chloride-free Ringer solution (35). Mice with a black coat color failed to respond to chloride-free Ringer solution, whereas agouti mice from the colony had a robust response. It was determined that mice with the black coat color that did not possess a chloride-free response had predominantly C57BL/6 alleles over a 4-cM region distal to agouti (35). To identify a possible C57BL/6 allele that regulated chloride-free response, pure C57BL/6J and 129/SvJ strains were studied. Surprisingly, pure C57BL/6J mice possessed a large chloride-free response, and pure 129/SvJ mice lacked this response (34). It is unclear what factors may be responsible for this difference, although heterogeneity of the strains used in the original study may account for these observed results. We have continued to use the pure C57BL/6J mice for these pharmacological studies because they have exhibited very reproducible responses to chloride-free Ringer solution. In these mice, the chloride-free response appears to be regulated by an NO-dependent pathway because pharmacological inhibition of iNOS activity significantly blocks this mode of chloride transport.
Similar results are obtained when C57BL/6J mice are treated with
the glucocorticoid dexamethasone. Dexamethasone has been shown to
downregulate the production of iNOS (4, 32, 33). We have substantiated
this property of dexamethasone in our system by showing reduced
staining of immunoreactive iNOS in lung sections from
dexamethasone-treated mice. Although ion transport properties of lower
airway and nasal epithelium may vary, this experiment substantiates the
in vivo loss of iNOS expression in response to dexamethasone treatment.
To further verify that the effects of dexamethasone on iNOS expression
are consistent in airway epithelium, NO production from the human
trachea epithelial cell line 9/HTEo cells treated with LPS was
examined in the presence and absence of dexamethasone.
Dexamethasone-treated cells had significantly reduced NO production
compared with control cells. Notably, Robbins et al. (28) have shown
that iNOS is expressed in the mouse lung epithelial cell line LA-4 in
response to various cytokines and that its expression is blocked by
dexamethasone. In this study, we compared the effects of dexamethasone
on ion transport regulation in murine nasal epithelium with the effects
of agents that directly influence the NO and cGMP pathways. Although
experiments with dexamethasone are difficult to interpret because of
the pleiotropic effects of corticosteroids, the inhibition of the
chloride-free response is consistent with the known ability of
dexamethasone to inhibit NO production. Additionally,
glucocorticoids have been shown to be quite effective in downregulating
iNOS production and thus may represent a natural regulator of iNOS
expression and the subsequent downstream effects such as ion transport regulation.
Finally, we examined NOS2(/
) mice that have been
backcrossed onto a C57BL/6J background for a minimum of seven
generations and compared their chloride-free responses with those of
wild-type C57BL/6J mice. Consistent with the previous studies
with SMT and dexamethasone, the NOS2(
/
) mice have a
significantly reduced chloride-free response compared with the robust
responses from C57BL/6J mice. These mice, however, have normal
CFTR activity as indicated by responses to forskolin (data not shown).
These data suggest that CFTR activity is an important component of the chloride-free response but is not the major contributor to
basal chloride transport. As a comparison to the B6129F2
mice, chloride-free responses in NOS2(
/
) mice were tested
in the presence of L-arginine. Unlike the B6129F2 mice,
L-arginine had no effect on
chloride transport in the NOS2(
/
) mice, demonstrating
that constitutively expressed iNOS is an important factor in
transepithelial chloride transport.
These studies clearly show the involvement of NO-dependent signaling
pathways in regulating the chloride-free response. These methods are
less conclusive, however, in understanding the role of NO in regulating
basal sodium transport. Acute inhibition of iNOS activity by the
addition of SMT clearly indicates an increase in amiloride-sensitive
sodium transport. We have previously shown that cGMP plays an important
function in regulating amiloride-sensitive sodium transport in airway
epithelial cells (18), which is consistent with other reports showing a
similar role for cGMP in renal and vascular systems (24, 30). The
effects seen with SMT are expected given the stimulation of guanylate
cyclase activity by NO. Also as expected on the basis of results with
SMT, mice treated with dexamethasone demonstrated an increased
hyperpolarization of baseline nasal TEPD. However, this increase does
not appear to be solely the result of an increase in
amiloride-sensitive sodium transport. This finding may be an artifact
associated with the many systems that are influenced by dexamethasone
and may not reflect strict NO regulation of sodium
transport. Likewise, NOS2(/
) mice have baseline nasal TEPD values that are identical to those in the C57BL/6J mice. However, both the wild-type C57BL/6J and
NOS2(
/
) mice have elevated baseline values compared with
the B6129F2 mice and compared with other strains tested (data not
shown). Therefore, any elevation in baseline TEPD as a result of a lack
of iNOS expression in the NOS2(
/
) mice may be masked by a
strain variation that leads to elevated baselines in the
C57BL/6J mice. Despite the ambiguity of the effects of
dexamethasone and the deletion of iNOS expression in the
NOS2(
/
) mice on baseline TEPD, our conclusion that NO
plays a vital role in the downregulation of transepithelial sodium
reabsorption is supported by two recent reports that show that NO
inhibits amiloride-sensitive sodium absorption in rat type II alveolar
epithelial cells (12, 15).
Given recent observations that iNOS levels are reduced in CF airway epithelium (19, 26), a clearer understanding of how NO influences various aspects of lung physiology is needed. With evidence that a lack of NO will further compound electrolyte transport problems associated with CF, perhaps therapeutic options that restore these disrupted pathways can be identified and used to augment existing attempts to correct CFTR function.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant from the Cystic Fibrosis Foundation.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests: T. J. Kelley, Dept. of Pediatrics, Case Western Reserve Univ., 8th floor BRB, 10900 Euclid Ave., Cleveland, OH 44106-4948 (E-mail: tjk12{at}po.cwru.edu).
Received 9 July 1998; accepted in final form 16 November 1998.
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