Alterations in airway ion transport in NKCC1-deficient mice

B. R. Grubb, A. J. Pace, E. Lee, B. H. Koller, and R. C. Boucher

Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599-7248


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion compensated for reduced Cl- 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, Slc12a2Delta 1065-1137, resulted in animals carrying a deletion mutation, and the other plasmid, Slc12a2Delta 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<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 0.4 H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, and 25 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. In experiments in which the tissue was bathed with a nominally Cl--free HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> buffer (referred to as zero Cl- 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<UP><SUB>3</SUB><SUP>−</SUP></UP> buffer replaced the Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. The pH of all these buffers was 7.4 when gassed with 95% O2-5% CO2 during the experiment. For the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free buffer, 10 mM HEPES buffer titrated to pH 7.4 with 1 M Tris base replaced the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. This buffer was gassed with 100% O2. All preparations contained 5 mM glucose in the basolateral and 5 mM mannitol in the apical solution.

Amiloride (10-4 M apical addition) was used to block electrogenic Na+ absorption. Forskolin (10-5 M apical) and UTP (10-4 M apical) were used to induce anion secretion via an increase in cAMP and Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>, respectively. Selected drugs were used in an attempt to characterize basolateral anion entry paths. Bumetanide (10-4 M), an inhibitor of NKCC1, was added to the basolateral bath. DIDS (10-3 M) was added to the basolateral bath to block the Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger. Acetazolamide (10-3 M bilaterally) was used to block the action of carbonic anhydrase, the enzyme that catalyzes the endogenous production of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. All drugs were purchased from Sigma with the exception of UTP (Amersham Pharmacia Biotech) and DIDS (Molecular Probes).

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-2 · 4 min-1).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


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Fig. 1.   In situ hybridization analysis of Na+-K+-2Cl- (NKCC1) mRNA expression in neonatal (A and B) and adult (C and D) trachea of wild-type mice. A and C: tracheal sections hybridized with a 35S-labeled NKCC1 antisense probe revealed no NKCC1 binding in adult trachea and intense NKCC1 binding on the epithelial cells lining the apical side of the neonatal tissue (arrows). B and D: neonatal and adult tracheal sections hybridized with a 35S-labeled NKCC1 sense probe show little background binding. Bars, 70 µm.

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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Fig. 2.   Adult trachea. Basal, amiloride-sensitive, and postamiloride short-circuit current (Isc) was measured in normal (open bars) and NKCC1-/- (solid bars) adult trachea.



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Fig. 3.   Recorder traces of normal (A) and NKCC1-/- (B) adult trachea. There was substantial variability, especially in the amiloride (Amil) response in both groups of tissue. Amiloride responses in these traces are somewhat less than the mean response (Fig. 2) seen in both groups. C: response to forskolin (Forsk), UTP, and bumetanide (Bumet) of adult normal (open bars) and NKCC1-/- (solid bars) trachea. For each bar, n = 5. D: effect of solution replacement protocols on the mean UTP response of the adult trachea (open bars normal, closed bars NKCC1-/-). KRB, bilateral Krebs-Ringer HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> buffer (n = 5, both groups); 0 Cl-, Cl--free buffer on the basolateral side only (n = 4 normal, n = 3 NKCC1-/-); 0 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free buffer on the basolateral side only (n = 6 normal, n = 5 NKCC1-/-); 0 Na+, Na+-free buffer on the basolateral side only (n = 6 normal, n = 5 NKCC1-/-). *P <=  0.05 compared with normal tissue in the indicated buffer. *+P <=  0.05 compared with NKCC1-/-tissue in KRB. **P 0.05 <=  compared with normal tissue in KRB.

Several solution replacement protocols were carried out on tracheae from adult normal and NKCC1-/- mice in an attempt to characterize the ions required for the UTP-induced Isc. Data are shown for mean UTP responses because this parameter was most informative in discriminating between NKCC1-/- and normal tissue. Removal of Cl- from the basolateral bath significantly reduced the UTP mean response in the normal preparations but had no effect on the mean UTP response in the NKCC1-/- preparations (Fig. 3D). In the basolateral Cl--free buffer, the magnitude of the mean UTP responses did not differ between the two genotypes.

Basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> removal had no effect on the mean UTP response in the normal tracheae, whereas HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> removal significantly reduced the mean UTP response in the NKCC1-/- tissue compared with NKCC1-/- tissue in KRB (Fig. 3D). In HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free (basolateral) buffer, the -/- tissues exhibited mean UTP responses that were reduced ~7.5-fold compared with normal tissues in this buffer.

An additional group of tissue was studied in basolateral Na+-free buffer. This protocol significantly reduced the magnitude of the mean UTP response in the normal tissue compared with the response in KRB. A similar pattern was seen for the NKCC1-/- tissue (Fig. 3D). In basolateral Na+-free buffer, the -/- tissue exhibited a significantly attenuated mean UTP response compared with the normal tissue incubated in the same buffer.

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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Fig. 4.   Adult bronchi. A: basal, amiloride-sensitive, and postamiloride Isc in the bronchi of normal (open bars) and NKCC1-/- (closed bars) adults. For each group, n = 5. B: change in Isc in response to forskolin, UTP, and bumetanide in adult bronchi. For each group, n = 5. *P <=  0.05 bumetanide response of NKCC1-/-compared with normal. C: change in mean Isc integrated over a 4-min period in response to UTP. **P <=  0.01 compared with mean UTP normal bronchi.

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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Fig. 5.   Neonatal trachea. A: basal, amiloride-sensitive, and postamiloride Isc responses. B: change in Isc in response to the drugs indicated. For normal and NKCC1-/-, n = 21 and 17, respectively, for all but the UTP and bumetanide responses, where n = 7 for both genotypes. **P <=  0.01 compared with normal neonatal trachea for the treatments indicated.

Because the neonatal normal tracheae responded to bumetanide, whereas the normal adult tracheae did not, we investigated the relationship between bumetanide sensitivity and age of the mouse. Although there was scatter in the data, there was a highly significant negative correlation between tracheal bumetanide sensitivity (post-UTP) and the age of the mouse (Fig. 6). In this group of mice, bumetanide sensitivity was lost in mice 4-6 wk of age. However, occasionally we observed a bumetanide-sensitive response in the tracheae of mice 6 mo or older.


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Fig. 6.   Effect of age of mouse on the magnitude of the post-UTP tracheal Isc sensitivity to bumetanide (bumetanide-sensitive Isc); n = 4 at each time point, r = 0.95, P <=  0.01.

Because NKCC1 appeared to play a greater role in anion secretion in neonatal preparations compared with those from the adult mouse, we used various drug protocols to determine the anion secretory mechanisms mediating the UTP response in the neonatal tracheae. Acetazolamide pretreatment had no significant effect on the mean UTP response (response integrated over a 4-min period) in the normal mice, whereas this treatment significantly attenuated the UTP response in the NKCC1-/- preparations (Fig. 7A). DIDS alone (basolateral) failed to significantly alter the mean UTP response in either genotype (Fig. 7A). A combination of DIDS and acetazolamide significantly reduced the mean UTP response in both genotypes (Fig. 7A). Indeed, in the NKCC1-/- tissue, a combination of both drugs abolished the mean UTP response. In contrast, in the normal preparations, the UTP response after addition of the two drugs remained significantly greater than zero (Fig. 7A).


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Fig. 7.   Effect of various drug protocols on change in UTP-induced mean Isc in neonatal trachea. No Rx, no treatment; Acet, acetazolamide. Open bars, normal trachea; closed bars, NKCC1-/- trachea. A: n = 7 for No Rx for both genotypes; n = 5 and n = 6 for Acet normal and NKCC1-/-, respectively; n = 6 and n = 8 for DIDS normal and NKCC1-/-, respectively; and n = 5 for Acet + DIDS for both genotypes. B: n = 7 for Bumet normal; n = 5 for Acet+Bumet normal; n = 5 for DIDS+bumet; and n = 4 for Acet+DIDS+Bumet normal and NKCC1-/-. A: +P <=  0.05 for NKCC1-/-No Rx. *P <=  0.05 for normal preparations of No Rx. B: +P <=  0.05 for No Rx for NKCC1-/-(see A). *P <=  0.05 for indicated bar from bumetanide-treated normal preparations. **P < 0.05 compared with normal No Rx (see A).

In the normal neonatal tracheae, bumetanide treatment decreased the basal Isc (Delta Isc -19.4 ± 3.1 µA · cm-2, n = 7), whereas this drug had no effect on the NKCC1-/- tissue. Pretreatment with bumetanide significantly decreased the mean UTP response in the normal preparations (Fig. 7B) compared with No Rx (no treatment) normals (Fig. 7A). Furthermore, when normal neonatal tracheae were treated with either bumetanide plus acetzolamide or bumetanide plus DIDS (Fig. 7B), the UTP response was significantly attenuated compared with the UTP response of the untreated normal preparations (see Fig. 7A, No Rx).

In a final protocol, both normal and NKCC1-/- preparations were treated with a combination of acetazolamide, DIDS, and bumetanide. This protocol virtually eliminated the UTP mean response in both genotypes (Fig. 7B). Bumetanide, as expected, did not have an additional effect on the NKCC1-/- tissue, and the response of NKCC1-/- preparations did not change significantly from the protocol in which acetazolamide and DIDS were given. When exposed to the three-drug combination, the mean UTP response did not differ significantly between genotypes (Fig. 7B).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> production); DIDS (blocks the Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger)] to help characterize the functional activity of NKCC1 in our normal airway preparations. By comparing these data with data obtained from our NKCC1-/- 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).

Because the forskolin response in normal adult murine trachea has previously been shown to be attenuated by Cl- removal (14, 28), this response was thought to predominantly reflect Cl- secretion. Because the forskolin response did not differ between genotypes (Fig. 2C), it is likely that an alternative means of basolateral anion entry, possibly Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> anion exchange (AE) or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion, can maintain the forskolin response in the NKCC1-/- trachea. Several isoforms of the AE have been cloned, and in human airway tissue, mRNA for AE2 and AE3 have been molecularly identified (11). In equine tracheal epithelium, functional data suggest that in addition to NKCC1 entry, Cl- enters the basolateral side via a Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger coupled to a Na+/H+ exchanger (30).

Our data differ from a recently published study of another NKCC1-/- mouse (12), which, using cultured tracheal cells (presumably from adult mice), reported that the basal Isc was significantly reduced in the NKCC1-/- tracheal cells compared with normal cells. Furthermore, the NKCC1-/- cells in that report failed to respond to amiloride. That study (12) also reported a significantly attenuated forskolin response in the NKCC1-/- cells. We have previously demonstrated differences between cultured and freshly excised murine tracheae with regard to ion transport properties (14). Thus it appears that also, in this case, murine cultured airway epithelia do not accurately reflect the ion transport properties of freshly excised tissue.

In our preparations, stimulation with UTP resulted in a much larger increase in Isc than that observed after forskolin stimulation (Fig. 3). Other studies have found that the ATP-induced increase in Isc in the normal murine tracheae likely reflects a major Cl- secretory component (4, 5). Because UTP and ATP are equipotent for induction of a secretory response in murine trachea (7), both nucleotides likely act via the P2Y2 receptor to increase intracellular Ca2+. If the UTP response reflects Cl- secretion and NKCC1 plays a role in basolateral Cl- entry, then a difference in the UTP response between the normal and NKCC1-/- tissue would be expected. Clearly, the UTP response comprises at least two components, a fast, transient "peak" and a slower, more sustained response. In our freshly excised tracheal preparations, there was no difference in the magnitude of the peak UTP response between genotypes. We integrated the UTP response over a 4-min period (mean response) to quantitate the more sustained phase of the UTP response. This mean response was significantly reduced in the tracheae of the NKCC1-/- mice compared with that in the tracheae of the normal mice. There are a number of possibilities to explain the origin of the components of the UTP response; one is that much of the peak response may be generated by the secretion of intracellular Cl- (or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>), whereas much of the more sustained response may be generated by secretion of ions brought in by basolateral entry. In our freshly excised tracheal preparations, there was no difference in the magnitude of peak UTP response between genotypes, but the UTP-induced increase in Isc in the NKCC1-/- tracheae was more transient than in the control tissue (Fig. 3, A and B). When this response was integrated over a 4-min period, this mean response was significantly reduced in the tracheae of NKCC1-/- mice compared with that in the tracheae of the normal mice.

Although NKCC1 clearly plays a role in the UTP response, much of the response is preserved in the NKCC1-/- mice. To determine which ions were responsible for this response and basolateral entry mechanisms, additional experiments were undertaken. In the NKCC1-/- trachea, basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (but not Cl-) removal reduced the magnitude of the UTP response (Fig. 3D). Thus it appears that ~60% of the UTP response in NKCC1-/- tissue was dependent on the presence of basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. The remainder of the response may be due to endogenously produced HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Cl- secretion supported by basolateral entry via an AE is less likely, due to the absence of an effect of Cl- substitution on this response.

When Na+ was removed from the basolateral surface of the tracheal preparations, the magnitude of the mean UTP response in the tracheae of both genotypes was significantly reduced compared with respective preparations bathed in KRB (Fig. 3D). In the NKCC1-/- tissues, the magnitude of the response in the Na+-free media did not differ from that in basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free media. In normal tissues, absence of basolateral Na+ may attenuate the magnitude of the UTP response by several means: 1) inhibiting Cl- entry via NKCC1; 2) eliminating Cl- entry via Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange if this exchanger is coupled to the Na+/H+ exchanger; and 3) eliminating basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> entry via the Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter. The inhibition of the UTP-stimulated response in Na+-free buffer is similar to data reported by Devor et al. (10) in Calu-3 cells. Devor's study demonstrated that basolateral Na+ or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> removal markedly diminished the magnitude of the forskolin response, suggesting that the forskolin response reflected HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion, mediated via the Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter. Because no NKCC1 is expressed in the -/- tissue, the inhibition of the UTP response by Na+ removal cannot reflect the first mechanism (cited above). In addition, because basolateral Cl- removal did not attenuate the magnitude of the UTP response, the second mechanism appears to be unlikely. Therefore, the most likely scenario to explain the attenuated UTP response in the NKCC1-/- tissue when bathed in Na+-free media is that Na+ was necessary for Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter function (10).

Other studies have reported that cAMP stimulated not only Cl- secretion but also a residual anion flux (1), suggesting that airway epithelia may secrete HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. In canine tracheal epithelia, a large residual flux remained in epinephrine-treated tissue after furosemide treatment (31), the direction of which was consistent with HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion. Smith and Welsh (26) have shown that cAMP stimulated a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent Isc in canine and human airway epithelium and thus concluded that cAMP stimulates HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion in airway cells. This study also suggested that agents that increase intracellular Ca2+, including ATP, were found to stimulate a Isc in Cl--free buffer, which was suggested to also be HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion (26). The apical channel for conductive HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit is still uncertain, but a number of studies suggest that it may be secreted through CFTR (6, 9, 20, 26) or another independent apical anion conductance pathway, possibly the Ca2+-activated Cl- channel (6, 9, 20).

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.

The basal anion secretory function of the neonatal trachea was further characterized with a combination of three drugs: bumetanide, acetazolamide, and DIDS. As expected, pretreatment with bumetanide significantly decreased the magnitude of the basal Isc in the normal neonatal tracheae but had no effect on the NKCC1-/- tissue. Pretreatment of the tissues with DIDS (basolateral) failed to alter the basal Isc of the tracheae of either genotype (data not shown). Acetazolamide caused a small but significant inhibition in the basal Isc across the tracheae of both genotypes, but the inhibition in the NKCC1-/- tissue (Delta Isc -9.8 ± 2.8 µA · cm-2, n = 6) was approximately double that in normal tissue (Delta Isc -3.9 ± 0.8 µA · cm-2, n = 17). These data indicate that endogenous HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> production plays a small role in maintaining the basal Isc of the neonatal tracheae, and in the absence of NKCC1, this pathway appears to be upregulated.

We also examined the effect of these drugs on the UTP-stimulated Isc in the neonatal trachea (Fig. 7). Bumetanide significantly inhibited the mean UTP response in the normal tissue, but a substantial UTP response remained (Fig. 7B). Bumetanide had no effect on the UTP response in the NKCC1-/- tissue (Fig. 5), and the magnitude of the postbumetanide UTP response did not differ between the genotypes. Thus, as in normal adult airway, a significant fraction of the UTP response (about one-third) did not appear to be sustained by basolateral Cl- entry via NKCC1.

Acetazolamide significantly altered the magnitude of the UTP response only in the NKCC1-/- tissue, and DIDS was without effect on the UTP response of tissue of either genotype (Fig. 7A). However, pretreatment of the tissue with a combination of the two drugs significantly decreased the magnitude of the UTP response in both genotypes. In the normal tissue, this drug combination resulted in a 60% decrease in the magnitude of the UTP response, and in the NKCC1-/- tissue, the UTP response did not differ significantly from zero. The finding that, in combination, these drugs were additive suggests that two separate processes were responsible for the UTP secretory response. Unfortunately, the effect of DIDS is not specific, and studies have shown that it can inhibit both the Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters (25) and the AEs (23). Also, the inhibitory effect of DIDS on the AEs is variable and possibly isoform and species specific (23, 30). Therefore, with the data available, we cannot be certain which transport pathway(s) was blocked by DIDS. However, on the basis of the ion substitution data obtained from the adult -/- tracheae (UTP response depends on basolateral Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> but not Cl-) and the drug protocols for the neonatal tracheae (total inhibition of the UTP response by DIDS + acetazolamide), we speculate that the UTP response in the NKCC1-/- tracheae was likely maintained by a basolateral Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter and endogenous HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> production.

With the DIDS plus acetazolamide drug combination, the normal neonatal tissue still exhibited a significant response to UTP (Fig. 7A). This residual response in normal tissue seemed likely to reflect a Cl- secretory component (data from the adult normal trachea demonstrate that basolateral Cl- is necessary; Fig. 3), with basolateral entry mediated via NKCC1. Therefore, the effect of bumetanide combined with either DIDS or acetazolamide was tested on normal neonatal tissue (Fig. 7B). Both of these protocols significantly reduced the UTP response in normal tissue to approximately the same extent (a reduction of ~75%). A combination of acetazolamide, DIDS, and bumetanide reduced the UTP response in the normal neonatal tissue to zero. Therefore, in normal neonatal trachea the basolateral entry pathways needed to maintain the UTP response appear to reflect a combination of NKCC1, a DIDS-sensitive pathway (either Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter or AE), and endogenously produced HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>.

In conclusion, we have studied the airway epithelia of adult and neonatal mice in which the NKCC1 gene has been disrupted to gain insight into the importance of this protein in basal and stimulated anion secretion in airway epithelia. The airway epithelia of both the adult and neonatal NKCC1-/- mouse exhibited a transport defect when a vigorous anion secretory response was stimulated by UTP. Thus NKCC1 played a more dominant and rate-limiting role when the secretory response was rapid and of relatively large magnitude. In contrast, when the secretory response was of smaller magnitude (response to forskolin), the responses in the NKCC1-/- tissues were not attenuated, and the alternative basolateral anion entry mechanisms appeared capable of sustaining secretion. Our data suggest that secretion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> endogenously produced and basolaterally transported was most likely the mechanism by which the NKCC1-/- airway epithelia were able to sustain (albeit reduced) a secretory response to UTP. The absence of airway pathology in the NKCC1-/- pups (12), along with the lack of spontaneous airway disease in our older NKCC1-/- mice (unpublished observation), add support to the hypothesis that the mice are able to compensate for absence of the NKCC1 protein by other pathways.


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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