Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599-7248
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ABSTRACT |
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Airways of Na+-K+-2Cl
(NKCC1)-deficient mice (
/
) were studied in Ussing chambers to
determine the role of the basolateral NKCC1 in transepithelial anion
secretion. The basal short-circuit current (Isc)
of tracheae and bronchi from adult mice did not differ between
NKCC1
/
and normal mice, whereas NKCC1
/
tracheae from neonatal
mice exhibited a significantly reduced basal
Isc. In normal mouse tracheae, sensitivity to
the NKCC1 inhibitor bumetanide correlated inversely with the age of the
mouse. In contrast, tracheae from NKCC1
/
mice at all ages were
insensitive to bumetanide. The anion secretory response to forskolin
did not differ between normal and NKCC1
/
tissues. However, when
larger anion secretory responses were induced with UTP, airways from
the NKCC1
/
mice exhibited an attenuated response. Ion substitution
and drug treatment protocols suggested that HCO
secretion in
NKCC1
/
airway epithelia. The absence of spontaneous airway disease
or pathology in airways from the NKCC1
/
mice suggests that the
NKCC1 mutant mice are able to compensate adequately for absence of the
NKCC1 protein.
bumetanide; Na+-K+-2Cl
cotransporter; bicarbonate secretion; chloride secretion
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INTRODUCTION |
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IN ADDITION TO SODIUM
ABSORPTION, Cl secretion and the osmotically linked
water flow across airway epithelia may contribute to the maintenance of
the depth of the airway surface liquid optimal for mucociliary
clearance. For Cl
secretion to be generated across the
epithelial cell, there must be coordination between apical
Cl
exit and basolateral Cl
entry as well as
maintenance of an electrochemical driving force to induce anion
secretion. The cAMP-mediated apical Cl
channel (cystic
fibrosis transmembrane conductance regulator; CFTR) has been
intensively investigated over the last 10 yr in an attempt to determine
how a defect in this channel causes airway disease in cystic fibrosis.
In addition, the Ca2+-activated apical Cl
channel has been extensively studied and is a potential therapeutic target through which Cl
can be secreted when CFTR is
absent/nonfunctional (2, 9, 29).
In contrast, much less attention has been directed at the basolateral
Cl entry mechanisms. There are several Cl
transport mechanisms capable of moving Cl
across the
basolateral membrane and into the cell. However, it is generally
believed that the primary basolateral Cl
entry pathway in
airway epithelia is the
Na+-K+-2Cl
cotransporter (NKCC1)
(19). This transporter moves these ions electroneutrally
across the basolateral membrane, with the usual stoichiometry being 1 Na+:1 K+ and 2 Cl
(17). Bumetanide and other loop diuretics are effective at inhibiting NKCC1 (17) and thus have been used extensively
in investigating the physiology of this basolateral cotransport protein.
We have generated mice lacking functional NKCC1 (22) by gene targeting. Using a combination of ion substitution studies and bumetanide as well as other selected drugs, we have investigated the importance of NKCC1 in basal as well as stimulated anion secretory responses in freshly excised tracheae and bronchi from adult and neonatal mice.
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METHODS |
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Using two targeting plasmids, we have generated two
NKCC1-deficient mouse lines in which the Slc12a2 gene, which
codes for NKCC1, was disrupted in embryonic stem cell lines.
One of the targeting plasmids,
Slc12a21065-1137, resulted in animals
carrying a deletion mutation, and the other plasmid,
Slc12a2
506-621, resulted in animals carrying
a null allele (22). Interestingly, the phenotype of the
animals carrying either of these mutations appears identical
(22). Animals carrying the null allele were used in this
investigation. Mice were bred on a heterogeneous strain background
(C57BL/6J+DBA/2J) (22).
Adult mice studied were 4-12 mo of age. The normal mice
(homozygous normal, +/+) had a mean body mass of 24.2 ± 0.66 g (n = 6), whereas the littermate NKCC1 mutant mice
(referred to as NKCC1/
) had a significantly (P
0.01)
reduced body mass [18.l. ± 0.75 g (n = 5)] compared
with their normal littermates. (Not all animals used in this study were
weighed.) Animals were allowed access to food and water until they were
euthanized with 100% CO2.
The mouse pups studied remained with the mother until time of study.
The mean age of the pups studied was 10 days, excluding those used in
the study to correlate bumetanide sensitivity with age
(n = 28). The mean body mass of the normal
homozygous/heterozygous pups (5.67 ± 0.25g; n = 29) was significantly greater (P 0.01) than that of the
NKCC1
/
pups (3.9 ± 0.47 g; n = 15). The pups were
killed by decapitation. A piece of tail was obtained from each pup, and
genotype was determined by Southern blot (22) at a later
time. Thus these studies were done blinded with respect to genotype.
Details of the Ussing chamber setup have been previously published (14). Briefly, the adult tracheae were mounted on Ussing chambers having an exposed surface area of 0.025 cm2, whereas the bronchi (mainstem and occasionally secondary bronchi) and the neonatal tracheae were mounted on chambers with an exposed surface area of 0.014 cm2. All preparations were equilibrated for 30 min before the first bioelectric measurements were recorded. Electrical measurements were made under short-circuit conditions. Resistance was calculated by Ohm's law by measuring the short-circuit current (Isc) change in response to a constant voltage pulse (1 mV).
Solutions and drugs.
Unless otherwise stated, tissues were bathed bilaterally with
Krebs-Ringer bicarbonate (KRB) having the following composition (in
mM): 140 Na+, 120 Cl, 5.2 K+, 1.2 Mg2+, 1.2 Ca2+, 2.4 HPO
-free HCO
buffer in the text), 115 mM sodium gluconate
replaced the NaCl and MgSO4 (1.2 mM) replaced the
MgCl2 [6 mM calcium gluconate was added to overcome the
Ca2+-chelating effects of gluconate, which replaced
CaCl2 (1.2 mM)]. For the Na+-free
buffer, N-methyl-D-glucamine (NMDG) replaced the
NaCl, and an NMDG HCO
In situ hybridization. The probe preparation and in situ hybridizations were carried out as described previously (13).
Data calculation and statistics. All data are shown as means ± SE with the number of tissues indicated in parentheses. Data were compared by a Student's t-test if only two groups were being compared. If more than two groups were being compared, an analysis of variance and a Student-Newman-Keuls test were used for multiple comparisons among groups.
For the UTP data, both the peak UTP response and the mean UTP response were measured. The mean UTP response was the area of the UTP peak integrated (SigmaScan) over a 4-min period and was expressed as a current flux (nEq · cm ![]() |
RESULTS |
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Before physiological studies were performed, mRNA expression
studies of NKCC1 in the trachea and lung of the adult and neonatal mouse were carried out. Analysis of neonatal tracheal epithelia by in
situ hybridization revealed dense, homogeneous binding of NKCC1
antisense probe in the superficial epithelial layer (Fig. 1A). In contrast, in situ
hybridization analysis of NKCC1 mRNA expression in adult
tracheal epithelia revealed little or no tracheal expression of NKCC1
mRNA (Fig. 1C).
|
Adult tracheae.
In the trachea of the adult mouse, the basal
Isc, the amiloride-sensitive
Isc, and the residual Isc
(postamiloride) did not differ between the normal and the NKCC1/
preparations (Fig. 2). The pattern of
response to drugs is shown in Fig. 3. The
only consistent difference between genotypes was that the UTP response in the normal preparations was more sustained (Fig. 3A) than
that exhibited by the NKCC1
/
tissues (Fig. 3B). The peak
changes in Isc in response to apical forskolin
or UTP did not differ between the genotypes (Fig. 3C).
However, when the UTP response was integrated over a 4-min period, the
NKCC1
/
tracheae exhibited significantly smaller "UTP mean
response" (expressed as a current flux) than did normal tissue (Fig.
3D, KRB). Interestingly, virtually none of the adult
tracheae (either normal or NKCC1
/
) exhibited a significant response
to bumetanide post-UTP (Fig. 3). Likewise, bumetanide did not decrease
the basal Isc or the response to forskolin when
given preforskolin (data not shown).
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Adult bronchi.
The bronchi, like the tracheae, did not differ between genotypes with
respect to the basal Isc, amiloride-sensitive
Isc, or postamiloride Isc (Fig.
4A). The forskolin-stimulated
Isc also did not differ between the genotypes
(Fig. 4B). The peak UTP response did not differ
significantly between the genotypes (Fig. 4B), but again the
mean UTP response was significantly attenuated in the NKCC1/
bronchi (Fig. 4C). The magnitude of the UTP response (mean
and peak) of the bronchi was significantly less (P
0.01) compared with the tracheal response for each respective genotype. In
contrast to the tracheae, the normal bronchi responded to bumetanide with a significant attenuation in the UTP-stimulated
Isc, whereas the NKCC1
/
bronchi failed to
respond to this drug (Fig. 4B).
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Neonatal tracheae.
The trachea of neonatal mouse pups (mean age 10 days) was studied to
gain insight into the role of the basolateral NKCC1 in the
Cl secretory response of the airway epithelium as a
function of age. Unlike in the adult trachea, the basal
Isc of the NKCC1
/
tissue was significantly
reduced compared with tissue from normal pups (Fig.
5A). The magnitude of the
amiloride-sensitive Isc was smaller in neonatal
than adult trachea but did not differ between the two genotypes. The
postamiloride Isc was significantly reduced in
the NKCC1
/
neonatal tracheae compared with normals (Fig. 5A). The response to forskolin was small and did not differ
between the two genotypes (Fig. 5B). However, the peak
response to UTP (Fig. 5B) as well as the mean UTP response
(see Fig. 7A, No Rx) was significantly attenuated in the
NKCC1
/
neonatal preparations (Fig. 5B). Interestingly,
unlike the normal adult tracheae, the normal neonatal tracheae
exhibited a significant response to bumetanide (post-UTP), whereas the
NKCC1
/
tissue failed to respond to the drug (Fig. 5B).
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DISCUSSION |
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NKCC1 has been proposed as the principal basolateral
Cl entry mechanism in airway epithelia (19).
This basolateral cotransporter, coupled with basolateral K+
channels, Na+-K+-ATPase, and apical
Cl
channels, has been suggested to work in concert to
effect Cl
secretion in many secretory epithelia
(17).
The loop diuretics (furosemide and bumetanide) bind to NKCC1 to inhibit
its function. The magnitude of bumetanide binding appears to reflect
the number of functional transport proteins in the basolateral membrane
(16). Thus we have used this drug as well as others
[acetazolamide (blocks endogenous HCO/HCO
/
mice, we have obtained some insight into the role of NKCC1
in the basal and stimulated anion secretory response in murine airways.
In situ hybridization analyses revealed that the trachea of the
neonatal mouse expressed abundant mRNA encoding the cotransporter, while NKCC1 mRNA was not detected in the tracheae of adult mice (Fig.
1) (see also Ref. 24). In contrast to the adult tracheae, previous in situ studies revealed mRNA expression for NKCC1 in the
bronchi of adult mice (24). Our functional data
(bumetanide sensitivity) agreed with the in situ hybridization
data (Figs. 2, 4, and 5). We detected no bumetanide sensitivity
in the trachea of the normal adult mouse, whereas bumetanide was
effective in the neonatal tracheae and adult bronchi. Our data
demonstrated that bumetanide sensitivity in the trachea decreased with
age, and at ~6 wk of age the mouse trachea became refractory to loop diuretics (Fig. 6). However, it should be noted that we occasionally observed a bumetanide response in the tracheae of older mice (up to 6 mo). We and others have reported tracheal bumetanide (or furosemide)-sensitive Isc in mice older than 4 wk (14, 21, 27, 28). Consequently, there may be strain
differences in the age at which bumetanide sensitivity is lost, or
possibly some strains of mice do not lose tracheal bumetanide
sensitivity with age. Despite the absence of bumetanide sensitivity in
the normal adult tracheae, comparison of responses of wild-type and
NKCC1/
mice indicated that NKCC1 was functional in adult tracheae
(see below). Thus these data point out that the lack of a response to
bumetanide cannot be used to conclude that there is no functional NKCC1.
Adult trachea.
The adult NKCC1/
trachea exhibited no significant difference in the
basal Isc, amiloride-sensitive
Isc, or postamiloride Isc
compared with the pattern exhibited by trachea from the normal mouse
(Fig. 2). A substantial portion of the basal Isc
in the preparations of both genotypes was not amiloride sensitive and thus not likely due to Na+ absorption. We have previously
suggested that this "residual" (postamiloride)
Isc reflects anion secretion (14).
Adult bronchi.
The bioelectric properties of bronchi "missing" functional NKCC1
were very similar to those observed in the trachea. Only the mean UTP
response in the NKCC1/
bronchi was significantly reduced compared
with controls. However, a major difference between the tracheae and the
bronchi was that the normal tracheae failed to respond to bumetanide,
whereas the normal bronchi exhibited a significant bumetanide response
(Fig. 4). None of the NKCC1
/
tissues, as expected, responded to
bumetanide. However, despite the molecular and functional evidence
(bumetanide sensitivity) that NKCC1 is more abundant in adult bronchi
and tracheae, both the peak and the mean UTP responses were
significantly smaller in bronchi compared with tracheae for each
genotype. Haas et al. (18) also reported that there was a
greater abundance of NKCC1 protein in bronchi compared with tracheae
(canine), yet in the canine bronchi the magnitude of the stimulated
Cl
secretory response was less (3, 15).
Boucher and Larsen (3) have suggested that there is a
larger basolateral Cl
conductance in canine bronchi than
in trachea; thus Cl
entering via NKCC1 would be shunted
across the basolateral membrane, blunting the Cl
secretory response. It is possible that in mice a similar scenario may
explain why the magnitude of anion secretory responses was less in the
murine bronchi compared with the tracheae.
Neonatal trachea.
To further characterize the importance of the NKCC1 cotransporter in
the Cl secretory response of murine airway
epithelia, we investigated the basal and stimulated bioelectric
properties of neonatal trachea, which express abundant mRNA for the
cotransporter (Fig. 1) and a substantial response to bumetanide (Fig.
5). Unlike in the adult trachea, the basal and postamiloride
Isc were significantly reduced in the NKCC1
/
tracheae compared with normal neonatal tissue (Fig. 5). This finding
suggests that in normal neonatal tissue, NKCC1 is important for
supplying the Cl
to sustain the basal
Isc. The responses to forskolin were small and
did not differ between the two genotypes. However, in the neonatal
trachea, both the peak and the mean UTP responses were attenuated in
the NKCC1
/
tissue. Collectively, these data appear to reflect the
greater role of the cotransporter in normal neonatal tissue compared
with adult tissue.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institutes of Health Grants SCOR I-P50 HL-60280-01 and PPG 5-POI-HL-34322 and Cystic Fibrosis Foundation RDP RO26 (Project 14).
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. R. Grubb, Cystic Fibrosis/Pulmonary Research and Treatment Center, 7011 Thurston-Bowles Bldg., CB# 7248, Univ. of North Carolina, Chapel Hill, NC 27599-7248 (E-mail: bgrubb{at}med.unc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 19 January 2001; accepted in final form 19 March 2001.
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